Degradable clostridial toxins

ABSTRACT

The specification discloses Clostridial toxins or Clostridial toxin chimeras comprising an inactivation cleavage site, polynucleotide molecules encoding such toxins or chimeras, compositions comprising such toxins or chimeras, and method of producing such toxins or chimeras.

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 13/112,844, filed May20, 2011, now U.S. Pat. No. 8,512,992, which claims priority pursuant to35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No.61/346,578, filed on May 20, 2010, both incorporated entirely byreference.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). Clostridial toxins commerciallyavailable as pharmaceutical compositions include, BoNT/A preparations,such as, e.g., BOTOX® (Allergan, Inc., Irvine, Calif.),DYSPORT®/RELOXIN®, (Beaufour Ipsen, Porton Down, England), NEURONOX®(Medy-Tox, Inc., Ochang-myeon, South Korea) BTX-A (Lanzhou InstituteBiological Products, China) and XEOMIN® (Merz Pharmaceuticals, GmbH.,Frankfurt, Germany); and BoNT/B preparations, such as, e.g.,MYOBLOC™/NEUROBLOC™ (Elan Pharmaceuticals, San Francisco, Calif.). As anexample, BOTOX® is currently approved in one or more countries for thefollowing indications: achalasia, adult spasticity, anal fissure, backpain, blepharospasm, bruxism, cervical dystonia, essential tremor,glabellar lines or hyperkinetic facial lines, headache, hemifacialspasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and VII nerve disorder.

A Clostridial toxin treatment inhibits neurotransmitter release bydisrupting the exocytotic process used to secrete the neurotransmitterinto the synaptic cleft. There is a great desire by the pharmaceuticalindustry to expand the use of Clostridial toxin therapies beyond itscurrent myo-relaxant applications to treat sensory nerve-based ailments,such as, e.g., various kinds of chronic pain, neurogenic inflammationand urogenital disorders, as well as other disorders, such as, e.g.,pancreatitis. One approach that is currently being exploited to expandClostridial toxin-based therapies involves modifying a Clostridial toxinso that the modified toxin has an altered cell targeting capability fora non-Clostridial toxin target cell. This re-targeted capability isachieved by replacing a naturally-occurring targeting domain of aClostridial toxin with a targeting domain showing a preferential bindingactivity for a non-Clostridial toxin receptor present in anon-Clostridial toxin target cell. Such modifications to a targetingdomain result in a Clostridial toxin chimeric called a TargetedVesicular Exocytosis Modulating Protein (TVEMP) that is able toselectively bind to a non-Clostridial toxin receptor (target receptor)present on a non-Clostridial toxin target cell (re-targeted). AClostridial toxin chimeric with a targeting activity for anon-Clostridial toxin target cell can bind to a receptor present on thenon-Clostridial toxin target cell, translocate into the cytoplasm, andexert its proteolytic effect on the SNARE complex of the non-Clostridialtoxin target cell.

Clostridial toxin and Clostridial toxin chimeric therapies aresuccessfully used for many indications. Generally, administration of aClostridial toxin or Clostridial toxin chimeric is well tolerated.However, administration in some applications can be challenging becauseof the larger doses required to achieve a beneficial effect. Largerdoses can increase the likelihood that the toxin or Clostridial toxinchimeric may move through the interstitial fluids and the circulatorysystems, such as, e.g., the cardiovascular system and the lymphaticsystem, of the body, resulting in the undesirable dispersal of the toxinor Clostridial toxin chimeric to areas not targeted for treatment. Suchdispersal can lead to undesirable side effects, such as, e.g.,inhibition of neurotransmitter release in neurons not targeted for toxintreatment or paralysis of a muscle not targeted for treatment. Forexample, a patient administered a therapeutically effective amount of aBoNT/A treatment into the neck muscles for torticollis may developdysphagia because of dispersal of the toxin into the oropharynx. Thus,there remains a need for improved Clostridial toxins and/or Clostridialtoxin chimeras that are effective at the site of treatment, but havenegligible to minimal effects in areas not targeted for toxin treatment.

The growing clinical, therapeutic, and cosmetic use of Clostridialtoxins and Clostridial toxin chimeras in therapies requiring largerdoses necessitates the pharmaceutical industry to develop modifiedClostridial toxins and Clostridial toxin chimeras that are effective atthe target site of application, but reduce or prevent the possibleside-effects associated with the dispersal of the toxins to an unwantedlocation. The present specification provides novel modified Clostridialtoxins and Clostridial toxin chimeras that reduce or prevent unwantedside-effects associated with toxin dispersal into non-targeted areas.These and related advantages are useful for various clinical,therapeutic and cosmetic applications, such as, e.g., the treatment ofneuromuscular disorders, neuropathic disorders, eye disorders, pain,muscle injuries, headache, cardiovascular diseases, neuropsychiatricdisorders, endocrine disorders, cancers, otic disorders and hyperkineticfacial lines, as well as, other disorders where a Clostridial toxin or aClostridial toxin chimeric administration to a mammal can produce abeneficial effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a schematic of the current paradigm ofneurotransmitter release and Clostridial toxin intoxication in a centraland peripheral neuron. FIG. 1A shows a schematic for theneurotransmitter release mechanism of a central and peripheral neuron.The release process can be described as comprising two steps: 1) vesicledocking, where the vesicle-bound SNARE protein of a vesicle containingneurotransmitter molecules associates with the membrane-bound SNAREproteins located at the plasma membrane; and 2) neurotransmitterrelease, where the vesicle fuses with the plasma membrane and theneurotransmitter molecules are exocytosed. FIG. 1B shows a schematic ofthe intoxication mechanism for tetanus and botulinum toxin activity in acentral and peripheral neuron. This intoxication process can bedescribed as comprising four steps: 1) receptor binding, where aClostridial toxin binds to a Clostridial receptor system and initiatesthe intoxication process; 2) complex internalization, where after toxinbinding, a vesicle containing the toxin/receptor system complex isendocytosed into the cell; 3) light chain translocation, where multipleevents are thought to occur, including, e.g., changes in the internal pHof the vesicle, formation of a channel pore comprising the H_(N) domainof the Clostridial toxin heavy chain, separation of the Clostridialtoxin light chain from the heavy chain, and release of the active lightchain and 4) enzymatic target modification, where the activate lightchain of Clostridial toxin proteolytically cleaves its target SNAREsubstrate, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby preventingvesicle docking and neurotransmitter release.

FIG. 2 shows the domain organization of naturally-occurring Clostridialtoxins. The single-chain form depicts the amino to carboxyl linearorganization comprising an enzymatic domain, a translocation domain, anda binding domain. The di-chain loop region located between thetranslocation and enzymatic domains is depicted by the double SSbracket. This region comprises an endogenous di-chain loop proteasecleavage site that upon proteolytic cleavage with a naturally-occurringprotease, such as, e.g., an endogenous Clostridial toxin protease or anaturally-occurring protease produced in the environment, converts thesingle-chain form of the toxin into the di-chain form. Above thesingle-chain form, the H_(CC) region of the Clostridial toxin bindingdomain is depicted. This region comprises the β-trefoil domain whichcomprises in an amino to carboxyl linear organization an α-fold, a β4/β5hairpin turn, a β-fold, a β8/β9 hairpin turn, and a γ-fold.

FIGS. 3A and 3B show Clostridial toxins or Clostridial toxin chimeraswith a binding domain located at the amino terminus of the toxin. FIG.3A depicts the single-chain polypeptide form of a toxin or chimera withan amino to carboxyl linear organization comprising a binding element, atranslocation element, a di-chain loop region comprising an exogenousprotease cleavage site (P), and a therapeutic element. Upon proteolyticcleavage with a P protease, the single-chain form of the toxin orchimera is converted to the di-chain form. FIG. 3B depicts the singlepolypeptide form of a toxin or chimera with an amino to carboxyl linearorganization comprising a binding element, a therapeutic element, adi-chain loop region comprising an exogenous protease cleavage site (P),and a translocation element. Upon proteolytic cleavage with a Pprotease, the single-chain form of the toxin or chimera is converted tothe di-chain form.

FIGS. 4A, 4B, 4C and 4D show Clostridial toxins or Clostridial toxinchimeras with a binding domain located at the amino terminus of thetoxin. FIG. 4A depicts the single polypeptide form of a toxin or chimerawith an amino to carboxyl linear organization comprising a therapeuticelement, a di-chain loop region comprising an exogenous proteasecleavage site (P), a binding element, and a translocation element. Uponproteolytic cleavage with a P protease, the single-chain form of thetoxin or chimera is converted to the di-chain form. FIG. 4B depicts thesingle polypeptide form of a toxin or chimera with an amino to carboxyllinear organization comprising a translocation element, a di-chain loopregion comprising an exogenous protease cleavage site (P), a bindingelement, and a therapeutic element. Upon proteolytic cleavage with a Pprotease, the single-chain form of the toxin or chimera is converted tothe di-chain form. FIG. 4C depicts the single polypeptide form of atoxin or chimera with an amino to carboxyl linear organizationcomprising a therapeutic element, a binding element, a di-chain loopregion comprising an exogenous protease cleavage site (P), and atranslocation element. Upon proteolytic cleavage with a P protease, thesingle-chain form of the toxin or chimera is converted to the di-chainform. FIG. 4D depicts the single polypeptide form of a toxin or chimerawith an amino to carboxyl linear organization comprising a translocationelement, a binding element, a di-chain loop region comprising anexogenous protease cleavage site (P), and a therapeutic element. Uponproteolytic cleavage with a P protease, the single-chain form of thetoxin or chimera is converted to the di-chain form.

FIGS. 5A and 5B show Clostridial toxins or Clostridial toxin chimeraswith a binding domain located at the amino terminus of the toxin. FIG.5A depicts the single polypeptide form of a toxin or chimera with anamino to carboxyl linear organization comprising a therapeutic element,a di-chain loop region comprising an exogenous protease cleavage site(P), a translocation element, and a binding element. Upon proteolyticcleavage with a P protease, the single-chain form of the toxin isconverted to the di-chain form. FIG. 5B depicts the single polypeptideform of a toxin or chimera with an amino to carboxyl linear organizationcomprising a translocation element, a di-chain loop region comprising anexogenous protease cleavage site (P), a therapeutic element, and abinding element. Upon proteolytic cleavage with a P protease, thesingle-chain form of the toxin or chimera is converted to the di-chainform.

DETAILED DESCRIPTION

The present specification discloses modified Clostridial toxins andmodified Clostridial toxin chimeras that can be rapidly inactivated froman unwanted location or locations by exploiting the presence ofproteases present in interstitial fluids and circulatory systems, suchas, e.g., the cardiovascular system and the lymphatic system. This isbecause the modified Clostridial toxins and modified Clostridial toxinchimeras disclosed in the present specification comprise a proteasecleavage site for a protease present in an interstitial fluid and/or acirculatory system. The presence of such a protease cleavage site makesthe modified Clostridial toxin or modified Clostridial toxin chimericsusceptible to proteolytic cleavage by its cognate protease, whichrenders such modified toxins inactive. For example, in situations wherea Clostridial toxin or Clostridial toxin chimeric modified to comprise acleavage site for an extracellular matrix protease has diffused into theinterstitial fluid, this modified toxin or modified Clostridial toxinchimeric can be effectively cleaved by the cognate extracellular matrixprotease. As another example, in situations where a Clostridial toxin orClostridial toxin chimeric modified to comprise a cleavage site for ablood protease has diffused into the cardiovascular system, thismodified toxin or modified Clostridial toxin chimeric can be effectivelycleaved by the cognate blood protease. As yet another example, insituations where a Clostridial toxin or Clostridial toxin chimericmodified to comprise a cleavage site for a lymphatic protease hasdiffused into the lymphatic system, this modified toxin or modifiedClostridial toxin chimeric can be effectively cleaved by the cognatelymphatic protease. Thus utilizing a Clostridial toxin or Clostridialtoxin chimeric comprising a cleavage site(s) for proteases present theinterstitial fluid and/or circulatory system will lessen or remove suchClostridial toxin or Clostridial toxin chimeric from an unwantedlocation, thereby reducing or preventing the undesirable side-effectsassociated with the diffusion of a Clostridial toxin or Clostridialtoxin chimeric to an unwanted location.

Thus, aspects of the present specification provide a Clostridial toxincomprising an inactivation cleavage site located within an inactivationcleavage site region, wherein the inactivation cleavage site region islocated in the translocation domain or the H_(CN) binding subdomain.Such disclosed toxins can comprise a Clostridial toxin enzymatic domain,a Clostridial toxin translocation domain, a Clostridial toxin bindingdomain, a di-chain loop region comprising an exogenous protease cleavagesite, and an inactivation cleavage site located within an inactivationcleavage site region. Non-limiting examples of inactivation cleavagesites include Thrombin cleavage sites, Plasmin cleavage sites,Coagulation Factor VIIa cleavage sites, Coagulation Factor IXa cleavagesites, Coagulation Factor Xa cleavage sites, Coagulation Factor XIacleavage sites, Coagulation Factor XIIa cleavage sites, plasmakallikrein cleavage sites, protease-activated G protein-coupledreceptor-1 (PAR1) cleavage sites, PAR2 cleavage sites, PAR3 cleavagesites, PAR4 cleavage sites, Matrix Metalloproteinase-2 (MMP-2) cleavagesites, Matrix Metalloproteinase-9 (MMP-9) cleavage sites, Furin cleavagesites, urokinase-type Plasminogen activator (uPA) cleavage sites,tissue-type Plasminogen activator (tPA) cleavage sites, Tryptase-εcleavage sites, Mouse mast cell protease-7 (mMCP-7) cleavage sites,endothelin-converting enzyme-1 (ECE-1) cleavage sites, Kell blood groupcleavage sites, DPPIV cleavage sites, ADAM metallopeptidase withthrombospondin type 1 motif-13 (ADAMTS13) cleavage sites, and CathepsinL cleavage sites. The addition of the inactivation cleavage siteincreases the safety margin of the Clostridial toxin or Clostridialtoxin chimeric relative to the same or similar Clostridial toxin orClostridial toxin chimeric, but without the additional inactivationcleavage site.

Other aspects of the present specification provide a Clostridial toxinchimeric comprising a Clostridial toxin enzymatic domain, a Clostridialtoxin translocation domain, a non-Clostridial toxin binding domain, andan inactivation cleavage site located within an inactivation cleavagesite region, wherein the inactivation cleavage site region is located inthe translocation domain or the H_(CN) binding subdomain. Such disclosedtoxins can comprise a Clostridial toxin enzymatic domain, a Clostridialtoxin translocation domain, a non-Clostridial toxin binding domain, adi-chain loop region comprising an exogenous protease cleavage site, andan inactivation cleavage site located within an inactivation cleavagesite region. Non-limiting examples of inactivation cleavage sitesinclude Thrombin cleavage sites, Plasmin cleavage sites, CoagulationFactor VIIa cleavage sites, Coagulation Factor IXa cleavage sites,Coagulation Factor Xa cleavage sites, Coagulation Factor XIa cleavagesites, Coagulation Factor XIIa cleavage sites, plasma kallikreincleavage sites, protease-activated G protein-coupled receptor-1 (PAR1)cleavage sites, PAR 2 cleavage sites, PAR3 cleavage sites, PAR4 cleavagesites, Matrix Metalloproteinase-2 (MMP-2) cleavage sites, MatrixMetalloproteinase-9 (MMP-9) cleavage sites, Furin cleavage sites,urokinase-type Plasminogen activator (uPA) cleavage sites, tissue-typePlasminogen activator (tPA) cleavage sites, Tryptase-ε cleavage sites,Mouse mast cell protease-7 (mMCP-7) cleavage sites,endothelin-converting enzyme-1 (ECE-1) cleavage sites, Kell blood groupcleavage sites, DPPIV cleavage sites, ADAM metallopeptidase withthrombospondin type 1 motif-13 (ADAMTS13) cleavage sites, and CathepsinL cleavage sites. The addition of the inactivation cleavage siteincreases the safety margin of the Clostridial toxin or Clostridialtoxin chimeric relative to the same or similar Clostridial toxin orClostridial toxin chimeric, but without the additional inactivationcleavage site.

Other aspects of the present specification provide polynucleotidemolecules encoding a Clostridial toxin or a Clostridial toxin chimericdisclosed in the present specification. A polynucleotide moleculeencoding such a Clostridial toxin or a Clostridial toxin chimeric canfurther comprise an expression vector.

Other aspects of the present specification provide a compositioncomprising a Clostridial toxin or a Clostridial toxin chimeric disclosedin the present specification. A composition comprising such aClostridial toxin or a Clostridial toxin chimeric can be apharmaceutical composition. Such a pharmaceutical composition cancomprise, in addition to a modified Clostridial toxin disclosed in thepresent specification a pharmaceutical carrier, a pharmaceuticalcomponent, or both.

Other aspects of the present specification provide a method of producinga Clostridial toxin or Clostridial toxin chimeric disclosed in thepresent specification, the method comprising the step of expressing in acell a polynucleotide molecule encoding a Clostridial toxin orClostridial toxin chimeric disclosed in the present specification,wherein expression from the polynucleotide molecule produces the encodedClostridial toxin or Clostridial toxin chimeric. In other aspects, themethod comprises the steps of introducing into a cell a polynucleotidemolecule encoding a Clostridial toxin or Clostridial toxin chimericdisclosed in the present specification, and expressing thepolynucleotide molecule, wherein expression from the polynucleotidemolecule produces the encoded Clostridial toxin or Clostridial toxinchimeric.

Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). BoNTs possess approximately 35%amino acid identity with each other and share the same functional domainorganization and overall structural architecture. It is recognized bythose of skill in the art that within each type of Clostridial toxinthere can be subtypes which differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3, BoNT/A4, and BoNT/A5, with specific subtypes showing about 84%to 93% amino acid identity when compared to the BoNT/A subtype of SEQ IDNO: 1. As another example, there are presently five BoNT/B subtypes,BoNT/B1, BoNT/B2, BoNT/B3, BoNT/Bnp, and BoNT/Bbv, with specificsubtypes showing about 93% to 96% amino acid identity when compared tothe BoNT/B subtype of SEQ ID NO: 6. As yet another example, there arepresently three BoNT/E subtypes, BoNT/E1, BoNT/E2, and BoNT/E3, withspecific subtypes showing about 95% to 99% amino acid identity whencompared to the BoNT/E subtype of SEQ ID NO: 15. While all seven BoNTserotypes have similar structure and pharmacological properties, eachalso displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other Clostridia species, C. baratii and C. butyricum,produce toxins, BaNT and BuNT, which are similar to BoNT/F and BoNT/E,respectively.

Clostridial toxins are each translated as a single chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease (FIG.1). This cleavage occurs within the discrete di-chain loop regioncreated between two cysteine residues that form a disulfide bridge. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by the single disulfide bond and non-covalentinteractions between the two chains. The naturally-occurring proteaseused to convert the single chain molecule into the di-chain is currentlynot known. In some serotypes, such as, e.g., BoNT/A, thenaturally-occurring protease is produced endogenously by the bacteriaserotype and cleavage occurs within the cell before the toxin isreleased into the environment. However, in other serotypes, such as,e.g., BoNT/E, the bacterial strain appears not to produce an endogenousprotease capable of converting the single chain form of the toxin intothe di-chain form. In these situations, the toxin is released from thecell as a single-chain toxin which is subsequently converted into thedi-chain form by a naturally-occurring protease found in theenvironment.

Each mature di-chain molecule comprises three functionally distinctdomains: 1) an enzymatic domain located in the LC that includes ametalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC(H_(N)) that facilitates releaseof the LC from intracellular vesicles into the cytoplasm of the targetcell; and 3) a binding domain found within the carboxyl-terminal half ofthe HC (H_(C)) that determines the binding activity and bindingspecificity of the toxin to the receptor complex located at the surfaceof the target cell. D. B. Lacy and R. C. Stevens, Sequence Homology andStructural Analysis of the Clostridial Neurotoxins, J. Mol. Biol. 291:1091-1104 (1999). The H_(C) domain comprises two distinct structuralfeatures of roughly equal size, separated by an α-helix, designated theH_(CN) and H_(CC) subdomains. Table 1 gives approximate boundary regionsfor each domain and subdomain found in exemplary Clostridial toxins.

TABLE 1 Clostridial Toxin Reference Sequences and Regions SEQ IDDi-Chain H_(C) Toxin NO: LC Loop H_(N) H_(CN) α-Linker H_(CC) BoNT/A 1M1/P2-L429 C430-C454 I455-I873 I874-N1080 E1081-Q1091 S1092-L1296 BoNT/B6 M1/P2-M436 C437-C446 I447-I860 L861-S1067 Q1068-Q1078 S1079-E1291BoNT/C1 11 M1/P2-F436 C437-C453 R454-I868 N869-D1081 G1082-L1092Q1093-E1291 BoNT/D 13 M1/T2-V436 C437-C450 I451-I864 N865-S1069N1069-Q1079 I1080-E1276 BoNT/E 15 M1/P2-F411 C412-C426 I427-I847K848-D1055 E1056-E1066 P1067-K1252 BoNT/F 18 M1/P2-F428 C429-C445I446-I865 K866-D1075 K1076-E1086 P1087-E1274 BoNT/G 21 M1/P2-M435C436-C450 I451-I865 S866-N1075 A1076-Q1086 S1087-E1297 TeNT 22M1/P2-L438 C439-C467 I468-L881 K882-N1097 P1098-Y1108 L1109-D1315 BaNT23 M1/P2-L420 C421-C435 I436-I857 I858-D1064 K1065-E1075 P1076-E1268BuNT 24 M1/P2-F411 C412-C426 I427-I847 K848-D1055 E1056-E1066P1067-K1251

The binding, translocation, and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Although the applicants have no wish to be limited by thefollowing description, the intoxication mechanism can be described ascomprising at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) enzymatic targetmodification (FIG. 3). The process is initiated when the H_(C) domain ofa Clostridial toxin binds to a toxin-specific receptor system located onthe plasma membrane surface of a target cell. The binding specificity ofa receptor complex is thought to be achieved, in part, by specificcombinations of gangliosides and protein receptors that appear todistinctly comprise each Clostridial toxin receptor complex. Once bound,the toxin/receptor complexes are internalized by endocytosis and theinternalized vesicles are sorted to specific intracellular routes. Thetranslocation step appears to be triggered by the acidification of thevesicle compartment. This process seems to initiate two importantpH-dependent structural rearrangements that increase hydrophobicity andpromote formation di-chain form of the toxin. Once activated, lightchain endopeptidase of the toxin is released from the intracellularvesicle into the cytosol where it appears to specifically target one ofthree known core components of the neurotransmitter release apparatus.These core proteins, vesicle-associated membrane protein(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa(SNAP-25) and Syntaxin, are necessary for synaptic vesicle docking andfusion at the nerve terminal and constitute members of the solubleN-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE)family. BoNT/A and BoNT/E cleave SNAP-25 in the carboxyl-terminalregion, releasing a nine or twenty-six amino acid segment, respectively,and BoNT/C1 also cleaves SNAP-25 near the carboxyl-terminus. Thebotulinum serotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanustoxin, act on the conserved central portion of VAMP, and release theamino-terminal portion of VAMP into the cytosol. BoNT/C1 cleavessyntaxin at a single site near the cytosolic membrane surface. Theselective proteolysis of synaptic SNAREs accounts for the block ofneurotransmitter release caused by Clostridial toxins in vivo. The SNAREprotein targets of Clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli etal., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9)Trends Microbiol. 431-437, (2003).

The three-dimensional crystal structures of BoNT/A, BoNT/B and the H_(C)domain of TeNT indicate that the three functional domains of Clostridialneurotoxins are structurally distinct domains that are shared by allClostridial toxins. The HEXXH consensus motif of the light chain formsthe tetrahedral zinc binding pocket of the catalytic site located in adeep cleft on the protein surface that is accessible by a channel. Thestructure of the H_(N) and H_(C) domains consists primarily of 6-sheettopologies that are linked by a single α-helix. The cylindrical-shapedH_(N) domain comprises two long amphipathic α-helices that resemble thecoiled-coil motif found in some viral proteins. The H_(N) domain alsoforms a long unstructured loop called the ‘translocation belt,’ whichwraps around a large negatively charged cleft of the light chain thatblocks access of the zinc atom to the catalytic-binding pocket of activesite. The H_(C) domain comprises two distinct structural features ofroughly equal size that indicate function. The first, designated theH_(CN) domain, is located in the amino half of the H_(C) domain. TheH_(CN) domain forms a β-barrel, jelly-roll fold. The H_(CC) domain isthe second domain that comprises the H_(C) domain. Thiscarboxyl-terminal domain comprises a modified β-trefoil domain whichforms three distinct carbohydrate binding regions that resembles thecarbohydrate binding moiety found in many sugar-binding proteins, suchas, e.g., serum amyloid P, sialidase, cryia, insecticidal δ-endotoxinand lectins. Biochemical studies indicate that the β-trefoil domainstructure of the H_(CC) domain appears to mediate the binding tospecific carbohydrate containing components of the Clostridial toxinreceptor on the cell surface, see, e.g., Krzysztof Ginalski et al.,Structure-based Sequence Alignment for the Beta-Trefoil Subdomain of theClostridial Neurotoxin Family Provides Residue Level Information Aboutthe Putative Ganglioside Binding Site, 482(1-2) FEBS Lett. 119-124(2000). The H_(C) domain tilts away from the H_(N) domain exposing thesurface loops and making them accessible for binding. No contacts occurbetween the light chain and the H_(C) domain.

Aspects of the present specification provide, in part, a Clostridialtoxin. As used herein, the term “Clostridial toxin” refers to anyneurotoxin produced by a Clostridial toxin strain that can execute theoverall cellular mechanism whereby a Clostridial toxin intoxicates acell and encompasses the binding of a Clostridial toxin to a low or highaffinity receptor complex, the internalization of the toxin/receptorcomplex, the translocation of the Clostridial toxin light chain into thecytoplasm and the enzymatic modification of a Clostridial toxinsubstrate. A Clostridial toxin comprises a Clostridial toxin enzymaticdomain, a Clostridial toxin translocation domain, and a Clostridialtoxin binding domain. Exemplary Clostridial toxins include thoseproduced by a Clostridium botulinum, a Clostridium tetani, a Clostridiumbaratii and a Clostridium butyricum.

A Clostridial toxin includes, without limitation, naturally occurringClostridial toxin variants, such as, e.g., Clostridial toxin isoformsand Clostridial toxin subtypes; non-naturally occurring Clostridialtoxin variants, such as, e.g., conservative Clostridial toxin variants,non-conservative Clostridial toxin variants, and active Clostridialtoxin fragments thereof, or any combination thereof. As used herein, theterm “Clostridial toxin variant,” whether naturally-occurring ornon-naturally-occurring, refers to a Clostridial toxin that has at leastone amino acid change from the corresponding region of the disclosedreference sequences (Table 1) and can be described in percent identityto the corresponding region of that reference sequence. As non-limitingexamples, a BoNT/A variant of SEQ ID NO: 1 will have at least one aminoacid difference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 1;a BoNT/B variant of SEQ ID NO: 6 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 6;a BoNT/C1 variant of SEQ ID NO: 11 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 11;a BoNT/D variant of SEQ ID NO: 13 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 13;a BoNT/E variant of SEQ ID NO: 15 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 15;a BoNT/F variant of SEQ ID NO: 18 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 18;a BoNT/G variant of SEQ ID NO: 21 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 21;a TeNT variant c of SEQ ID NO: 22 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 22;a BaNT variant of SEQ ID NO: 23 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 23;and a BuNT variant of SEQ ID NO: 24 will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to the corresponding position(s) of SEQ ID NO: 24.

As used herein, the term “naturally occurring Clostridial toxin variant”refers to any Clostridial toxin produced without the aid of any humanmanipulation, including, without limitation, Clostridial toxin isoformsproduced from alternatively-spliced transcripts, Clostridial toxinisoforms produced by spontaneous mutation and Clostridial toxinsubtypes. Non-limiting examples of a Clostridial toxin isoform include,e.g., BoNT/A isoforms, BoNT/B isoforms, BoNT/C1 isoforms, BoNT/Disoforms, BoNT/E isoforms, BoNT/F isoforms, BoNT/G isoforms, TeNTisoforms, BaNT isoforms and BuNT isoforms. Non-limiting examples of aClostridial toxin subtype include, e.g., BoNT/A subtypes BoNT/A1,BoNT/A2, BoNT/A3, BoNT/A4, and BoNT/A5; BoNT/B subtypes BoNT/B1,BoNT/B2, BoNT/B3, BoNT/B bivalent and BoNT/B nonproteolytic; BoNT/C1subtypes BoNT/C1-1 and BoNT/C1-2; BoNT/E subtypes BoNT/E1, BoNT/E2, andBoNT/E3; BoNT/F subtypes BoNT/F1, BoNT/F2, and BoNT/F3; and BuNTsubtypes BuNT-1, and BuNT-2. Other non-limiting examples of aClostridial toxin subtype include, e.g., BoNT/A subtypes SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; BoNT/Bsubtypes SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQID NO: 10; BoNT/C1 subtypes SEQ ID NO: 11 and SEQ ID NO: 12; BoNT/Esubtypes SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; BoNT/Fsubtypes SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20; and BuNTsubtypes SEQ ID NO: 24 and SEQ ID NO: 25.

As used herein, the term “non-naturally occurring Clostridial toxinvariant” refers to any Clostridial toxin produced with the aid of humanmanipulation, including, without limitation, Clostridial toxins producedby genetic engineering using random mutagenesis or rational design andClostridial toxins produced by chemical synthesis. Non-limiting examplesof non-naturally occurring Clostridial toxin variants include, e.g.,conservative Clostridial toxin variants, non-conservative Clostridialtoxin variants, and active Clostridial toxin fragments.

As used herein, the term “conservative Clostridial toxin variant” refersto a Clostridial toxin that has at least one amino acid substituted byanother amino acid or an amino acid analog that has at least oneproperty similar to that of the original amino acid from the referenceClostridial toxin sequence (Table 1). Examples of properties include,without limitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative Clostridial toxin variant canfunction in substantially the same manner as the reference Clostridialtoxin on which the conservative Clostridial toxin variant is based, andcan be substituted for the reference Clostridial toxin in any aspect ofthe present specification. A conservative Clostridial toxin variant maysubstitute 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, or500 or more amino acids from the reference Clostridial toxin on whichthe conservative Clostridial toxin variant is based. A conservativeClostridial toxin variant can also substitute at least 5, 10, 15, 20, or25 contiguous amino acids from the reference Clostridial toxin on whichthe conservative Clostridial toxin variant is based. Non-limitingexamples of a conservative Clostridial toxin variant include, e.g.,conservative BoNT/A variants, conservative BoNT/B variants, conservativeBoNT/C1 variants, conservative BoNT/D variants, conservative BoNT/Evariants, conservative BoNT/F variants, conservative BoNT/G variants,conservative TeNT variants, conservative BaNT variants and conservativeBuNT variants.

As used herein, the term “non-conservative Clostridial toxin variant”refers to a Clostridial toxin in which 1) at least one amino acid isdeleted from the reference Clostridial toxin on which thenon-conservative Clostridial toxin variant is based; 2) at least oneamino acid added to the reference Clostridial toxin on which thenon-conservative Clostridial toxin is based; or 3) at least one aminoacid is substituted by another amino acid or an amino acid analog thatdoes not share any property similar to that of the original amino acidfrom the reference Clostridial toxin sequence (Table 1). Anon-conservative Clostridial toxin variant can function in substantiallythe same manner as the reference Clostridial toxin on which thenon-conservative Clostridial toxin variant is based, and can besubstituted for the reference Clostridial toxin in any aspect of thepresent specification. A non-conservative Clostridial toxin variant candelete one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids, five or more amino acids, and tenor more amino acids from the reference Clostridial toxin on which thenon-conservative Clostridial toxin variant is based. A non-conservativeClostridial toxin variant can add one or more amino acids, two or moreamino acids, three or more amino acids, four or more amino acids, fiveor more amino acids, and ten or more amino acids to the referenceClostridial toxin on which the non-conservative Clostridial toxinvariant is based. A non-conservative Clostridial toxin variant maysubstitute 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, or500 or more amino acids from the reference Clostridial toxin on whichthe non-conservative Clostridial toxin variant is based. Anon-conservative Clostridial toxin variant can also substitute at least5, 10, 15, 20, or 25 contiguous amino acids from the referenceClostridial toxin on which the non-conservative Clostridial toxinvariant is based. Non-limiting examples of a non-conservativeClostridial toxin variant include, e.g., non-conservative BoNT/Avariants, non-conservative BoNT/B variants, non-conservative BoNT/C1variants, non-conservative BoNT/D variants, non-conservative BoNT/Evariants, non-conservative BoNT/F variants, non-conservative BoNT/Gvariants, non-conservative TeNT variants, non-conservative BaNT variantsand non-conservative BuNT variants.

It is also envisioned that any of a variety of Clostridial toxinfragments can be useful in aspects of the present specification with theproviso that these active fragments can execute the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. Thus, aspects of this embodiment can include Clostridialtoxin fragments having a length of, e.g., at least 600, 700, 800, 900,1000, 1100, or at least 1200 amino acids. Other aspects of thisembodiment, can include Clostridial toxin fragments having a length of,e.g., at most 600, 700, 800, 900, 1000, 1100, or at most 1200 aminoacids.

It is also envisioned that any of a variety of Clostridial toxinfragments comprising the light chain can be useful in aspects of thepresent specification with the proviso that these light chain fragmentscan specifically target the core components of the neurotransmitterrelease apparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The light chains of Clostridial toxins are approximately420-460 amino acids in length and comprise a Clostridial toxin enzymaticdomain (Table 1). Research has shown that the entire length of aClostridial toxin light chain is not necessary for the enzymaticactivity of the Clostridial toxin enzymatic domain. As a non-limitingexample, the first eight amino acids of a BoNT/A light chain are notrequired for enzymatic activity. As another non-limiting example, thefirst eight amino acids of the TeNT light chain are not required forenzymatic activity. Likewise, the carboxyl-terminus of the light chainis not necessary for activity. As a non-limiting example, the last 32amino acids of the BoNT/A light chain are not required for enzymaticactivity. As another non-limiting example, the last 31 amino acids ofthe TeNT light chain are not required for enzymatic activity. Thus,aspects of this embodiment include a Clostridial toxin light chaincomprising a Clostridial toxin enzymatic domain having a length of,e.g., at least 350, 375, 400, 425, or 450 amino acids. Other aspects ofthis embodiment include a Clostridial toxin light chain comprising aClostridial toxin enzymatic domain having a length of, e.g., at most350, 375, 400, 425, or 450 amino acids.

It is also envisioned that any of a variety of Clostridial toxin H_(N)regions comprising a Clostridial toxin translocation domain can beuseful in aspects of the present specification with the proviso thatthese active fragments can facilitate the release of the LC fromintracellular vesicles into the cytoplasm of the target cell and thusparticipate in executing the overall cellular mechanism whereby aClostridial toxin proteolytically cleaves a substrate. The H_(N) regionsfrom the heavy chains of Clostridial toxins are approximately 410-430amino acids in length and comprise a Clostridial toxin translocationdomain (Table 1). Research has shown that the entire length of a H_(N)region from a Clostridial toxin heavy chain is not necessary for thetranslocating activity of the Clostridial toxin translocation domain.Thus, aspects of this embodiment can include Clostridial toxin H_(N)regions comprising a Clostridial toxin translocation domain having alength of, e.g., at least 350, 375, 400, or 425 amino acids. Otheraspects of this embodiment can include Clostridial toxin H_(N) regionscomprising Clostridial toxin translocation domain having a length of,e.g., at most 350, 375, 400, or 425 amino acids.

It is also envisioned that any of a variety of Clostridial toxin H_(C)regions comprising a Clostridial toxin binding domain can be useful inaspects of the present specification with the proviso that these activefragments can determine the binding activity and binding specificity ofthe toxin to the receptor complex located at the surface of the targetcell and facilitate the overall cellular mechanism whereby a Clostridialtoxin proteolytically cleaves a substrate. The H_(C) regions from theheavy chains of Clostridial toxins are approximately 400-440 amino acidsin length and comprise a binding domain (Table 1). Research has shownthat the entire length of a H_(C) region from a Clostridial toxin heavychain is not necessary for the binding activity of the Clostridial toxinbinding domain. Thus, aspects of this embodiment can include Clostridialtoxin H_(C) regions comprising a binding domain having a length of,e.g., at least 350, 375, 400, or 425 amino acids. Other aspects of thisembodiment can include Clostridial toxin H_(C) regions comprising abinding domain having a length of, e.g., at most 350, 375, 400, or 425amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art and from the teaching herein. Globalmethods align sequences from the beginning to the end of the moleculeand determine the best alignment by adding up scores of individualresidue pairs and by imposing gap penalties. Non-limiting methodsinclude, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTALW: Improving the Sensitivity of Progressive Multiple Sequence AlignmentThrough Sequence Weighting, Position-Specific Gap Penalties and WeightMatrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); anditerative refinement, see, e.g., Osamu Gotoh, Significant Improvement inAccuracy of Multiple Protein Sequence Alignments by Iterative Refinementas Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol.823-838 (1996). Local methods align sequences by identifying one or moreconserved motifs shared by all of the input sequences. Non-limitingmethods include, e.g., Match-box, see, e.g., Eric Depiereux and ErnestFeytmans, Match-Box: A Fundamentally New Algorithm for the SimultaneousAlignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992);Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting SubtleSequence Signals: A Gibbs Sampling Strategy for Multiple Alignment,262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle etal., Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics,: 1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93(22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302(1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32(5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6(1) BMC Bioinformatics 66 (2005).

The present specification describes various polypeptide variants whereone amino acid is substituted for another, such as, e.g., Clostridialtoxin variants, Clostridial toxin enzymatic domain variants, Clostridialtoxin translocation domain variants, Clostridial toxin binding domainvariants, non-Clostridial toxin binding domain variants, and proteasecleavage site variants. A substitution can be assessed by a variety offactors, such as, e.g., the physical properties of the amino acid beingsubstituted (Table 2) or how the original amino acid would tolerate asubstitution (Table 3). The selections of which amino acid can besubstituted for another amino acid in a polypeptide are known to aperson of ordinary skill in the art.

TABLE 2 Amino Acid Properties Property Amino Acids Aliphatic G, A, I, L,M, P, V Aromatic F, H, W, Y C-beta branched I, V, T Hydrophobic C, F, I,L, M, V, W Small polar D, N, P Small non-polar A, C, G, S, T Large polarE, H, K, Q, R, W, Y Large non-polar F, I, L, M, V Charged D, E, H, K, RUncharged C, S, T Negative D, E Positive H, K, R Acidic D, E Basic K, RAmide N, Q

TABLE 3 Amino Acid Substitutions Amino Acid Favored Substitution NeutralSubstitutions Disfavored substitution A G, S, T C, E, I, K, M, L, P, Q,R, V D, F, H, N, Y, W C F, S, Y, W A, H, I, M, L, T, V D, E, G, K, N, P,Q, R D E, N G, H, K, P, Q, R, S, T A, C, I, L, E D, K, Q A, H, N, P, R,S, T C, F, G, I, L, M, V, W, Y F M, L, W, Y C, I, V A, D, E, G, H, K, N,P, Q, R, S, T G A, S D, K, N, P, Q, R C, E, F, H, I, L, M, T, V, W, Y HN, Y C, D, E, K, Q, R, S, T, W A, F, G, I, L, M, P, V I V, L, M A, C, T,F, Y D, E, G, H, K, N, P, Q, R, S, W K Q, E, R A, D, G, H, M, N, P, S, TC, F, I, L, V, W, Y L F, I, M, V A, C, W, Y D, E, G, H, K, N, P, Q, R,S, T M F, I, L, V A, C, R, Q, K, T, W, Y D, E, G, H, N, P, S N D, H, SE, G, K, Q, R, T A, C, F, I, L, M, P, V, W, Y P — A, D, E, G, K, Q, R,S, T C, F, H, I, L, M, N, V, W, Y Q E, K, R A, D, G, H, M, N, P, S, T C,F, I, L, V, W, Y R K, Q A, D, E, G, H, M, N, P, S, T C, F, I, L, V, W, YS A, N, T C, D, E, G, H, K, P, Q, R, T F, I, L, M, V, W, Y T S A, C, D,E, H, I, K, M, N, P, F, G, L, W, Y Q, R, V V I, L, M A, C, F, T, Y D, E,G, H, K, N, P, Q, R, S, W W F, Y H, L, M A, C, D, E, G, I, K, N, P, Q,R, S, T, V Y F, H, W C, I, L, M, V A, D, E, G, K, N, P, Q, R, S, TMatthew J. Betts and Robert B. Russell, Amino Acid Properties andConsequences of Substitutions, pp. 289-316, In Bioinformatics forGeneticists, (eds Michael R. Barnes, Ian C. Gray, Wiley, 2003).

Thus, in an embodiment, a Clostridial toxin comprises a Clostridialtoxin enzymatic domain, a Clostridial toxin translocation domain, and aClostridial toxin binding domain. In an aspect of this embodiment, aClostridial toxin comprises a naturally occurring Clostridial toxinvariant, such as, e.g., a Clostridial toxin isoform or a Clostridialtoxin subtype. In another aspect of this embodiment, a Clostridial toxincomprises a non-naturally occurring Clostridial toxin variant, such as,e.g., a conservative Clostridial toxin variant, a non-conservativeClostridial toxin variant or an active Clostridial toxin fragment, orany combination thereof. In another aspect of this embodiment, aClostridial toxin comprises a Clostridial toxin enzymatic domain or anactive fragment thereof, a Clostridial toxin translocation domain or anactive fragment thereof, a Clostridial toxin binding domain or an activefragment thereof, or any combination thereof. In other aspects of thisembodiment, a Clostridial toxin can comprise a BoNT/A, a BoNT/B, aBoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a TeNT, a BaNT, or aBuNT.

In another embodiment, a hydrophobic amino acid at one particularposition in the polypeptide chain of the Clostridial toxin can besubstituted with another hydrophobic amino acid. Examples of hydrophobicamino acids include, e.g., C, F, I, L, M, V and W. In another aspect ofthis embodiment, an aliphatic amino acid at one particular position inthe polypeptide chain of the Clostridial toxin can be substituted withanother aliphatic amino acid. Examples of aliphatic amino acids include,e.g., A, I, L, P, and V. In yet another aspect of this embodiment, anaromatic amino acid at one particular position in the polypeptide chainof the Clostridial toxin can be substituted with another aromatic aminoacid. Examples of aromatic amino acids include, e.g., F, H, W and Y. Instill another aspect of this embodiment, a stacking amino acid at oneparticular position in the polypeptide chain of the Clostridial toxincan be substituted with another stacking amino acid. Examples ofstacking amino acids include, e.g., F, H, W and Y. In a further aspectof this embodiment, a polar amino acid at one particular position in thepolypeptide chain of the Clostridial toxin can be substituted withanother polar amino acid. Examples of polar amino acids include, e.g.,D, E, K, N, Q, and R. In a further aspect of this embodiment, a lesspolar or indifferent amino acid at one particular position in thepolypeptide chain of the Clostridial toxin can be substituted withanother less polar or indifferent amino acid. Examples of less polar orindifferent amino acids include, e.g., A, H, G, P, S, T, and Y. In a yetfurther aspect of this embodiment, a positive charged amino acid at oneparticular position in the polypeptide chain of the Clostridial toxincan be substituted with another positive charged amino acid. Examples ofpositive charged amino acids include, e.g., K, R, and H. In a stillfurther aspect of this embodiment, a negative charged amino acid at oneparticular position in the polypeptide chain of the Clostridial toxincan be substituted with another negative charged amino acid. Examples ofnegative charged amino acids include, e.g., D and E. In another aspectof this embodiment, a small amino acid at one particular position in thepolypeptide chain of the Clostridial toxin can be substituted withanother small amino acid. Examples of small amino acids include, e.g.,A, D, G, N, P, S, and T. In yet another aspect of this embodiment, aC-beta branched amino acid at one particular position in the polypeptidechain of the Clostridial toxin can be substituted with another C-betabranched amino acid. Examples of C-beta branched amino acids include,e.g., I, T and V.

In another embodiment, a Clostridial toxin comprises a BoNT/A. In anaspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domain,a BoNT/A translocation domain, and a BoNT/A binding domain. In anotheraspect of this embodiment, a BoNT/A comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In another aspect ofthis embodiment, a BoNT/A comprises a naturally occurring BoNT/Avariant, such as, e.g., a BoNT/A isoform or a BoNT/A subtype. In anotheraspect of this embodiment, a BoNT/A comprises a naturally occurringBoNT/A variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO: 5, such as, e.g., a BoNT/A isoform or a BoNT/A subtype.In still another aspect of this embodiment, a BoNT/A comprises anon-naturally occurring BoNT/A variant, such as, e.g., a conservativeBoNT/A variant, a non-conservative BoNT/A variant or an active BoNT/Afragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/A comprises a non-naturally occurring BoNT/A variantof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5, such as, e.g., a conservative BoNT/A variant, a non-conservativeBoNT/A variant, an active BoNT/A fragment, or any combination thereof.In yet another aspect of this embodiment, a BoNT/A comprises a BoNT/Aenzymatic domain or an active fragment thereof, a BoNT/A translocationdomain or an active fragment thereof, a BoNT/A binding domain or anactive fragment thereof, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/A comprising a BoNT/A enzymatic domainSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5,or an active fragment thereof, a BoNT/A translocation domain of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or anactive fragment thereof, a BoNT/A binding domain of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or an activefragment thereof, or any combination thereof.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or at most70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most 95%to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:5. In yet other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500non-contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, orSEQ ID NO: 5. In still other aspects of this embodiment, a BoNT/Acomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, orSEQ ID NO: 5.

In another embodiment, a Clostridial toxin comprises a BoNT/B. In anaspect of this embodiment, a BoNT/B comprises a BoNT/B enzymatic domain,a BoNT/B translocation domain, and a BoNT/B binding domain. In anotheraspect of this embodiment, a BoNT/B comprises SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In another aspect ofthis embodiment, a BoNT/B comprises a naturally occurring BoNT/Bvariant, such as, e.g., a BoNT/B isoform or a BoNT/B subtype. In anotheraspect of this embodiment, a BoNT/B comprises a naturally occurringBoNT/B variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10, such as, e.g., a BoNT/B isoform or a BoNT/Bsubtype. In still another aspect of this embodiment, a BoNT/B comprisesa non-naturally occurring BoNT/B variant, such as, e.g., a conservativeBoNT/B variant, a non-conservative BoNT/B variant, an active BoNT/Bfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/B comprises a non-naturally occurring BoNT/B variantof SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:10, such as, e.g., a conservative BoNT/B variant, a non-conservativeBoNT/B variant, an active BoNT/B fragment, or any combination thereof.In yet another aspect of this embodiment, a BoNT/B comprises a BoNT/Benzymatic domain or an active fragment thereof, a BoNT/B translocationdomain or active fragment thereof, a BoNT/B binding domain or activefragment thereof, or any combination thereof. In yet another aspect ofthis embodiment, a BoNT/B comprises a BoNT/B enzymatic domain of SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, oractive fragment thereof, a BoNT/B translocation domain of SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or activefragment thereof, a BoNT/B binding domain of SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or active fragmentthereof, or any combination thereof.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 102; or at most70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most 95%to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:10. In yet other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500non-contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, orSEQ ID NO: 10. In still other aspects of this embodiment, a BoNT/Bcomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, orSEQ ID NO: 10.

In another embodiment, a Clostridial toxin comprises a BoNT/C1. In anaspect of this embodiment, a BoNT/C1comprises a BoNT/C1 enzymaticdomain, a BoNT/C1 translocation domain, and a BoNT/C1 binding domain. Inanother aspect of this embodiment, a BoNT/C1 comprises SEQ ID NO: 11 orSEQ ID NO: 12. In another aspect of this embodiment, a BoNT/C1 comprisesa naturally occurring BoNT/C1 variant, such as, e.g., a BoNT/C1 isoformor a BoNT/C1 subtype. In another aspect of this embodiment, a BoNT/C1comprises a naturally occurring BoNT/C1 variant of SEQ ID NO: 11 or SEQID NO: 12, such as, e.g., a BoNT/C1 isoform or a BoNT/C1 subtype. Instill another aspect of this embodiment, a BoNT/C1 comprises anon-naturally occurring BoNT/C1 variant, such as, e.g., a conservativeBoNT/C1 variant, a non-conservative BoNT/C1 variant, an active BoNT/C1fragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/C1 comprises a non-naturally occurring BoNT/C1variant of SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., a conservativeBoNT/C1 variant, a non-conservative BoNT/C1 variant, an active BoNT/C1fragment, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/C1 comprises a BoNT/C1 enzymatic domain or activefragment thereof, a BoNT/C1 translocation domain or active fragmentthereof, a BoNT/C1 binding domain, active fragment thereof, or anycombination thereof. In yet another aspect of this embodiment, a BoNT/C1comprises a BoNT/C1 enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12,or active fragment thereof, a BoNT/C1 translocation domain of SEQ ID NO:11 or SEQ ID NO: 12, or active fragment thereof, a BoNT/C1 bindingdomain of SEQ ID NO: 11 or SEQ ID NO: 12, or active fragment thereof, orany combination thereof.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 11or SEQ ID NO: 12; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to SEQ ID NO: 11 or SEQ ID NO: 12. In yetother aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,100, 200, 300, 400, or 500 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 11 or SEQ ID NO:12; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 11 or SEQ ID NO: 12. In still otheraspects of this embodiment, a BoNT/C1 comprises a polypeptide having,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 3; at most 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 11 orSEQ ID NO: 12.

In another embodiment, a Clostridial toxin comprises a BoNT/D. In anaspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domain,a BoNT/D translocation domain, and a BoNT/D binding domain. In anotheraspect of this embodiment, a BoNT/D comprises SEQ ID NO: 13 or SEQ IDNO: 14. In another aspect of this embodiment, a BoNT/D comprises anaturally occurring BoNT/D variant, such as, e.g., a BoNT/D isoform or aBoNT/D subtype. In another aspect of this embodiment, a BoNT/D comprisesa naturally occurring BoNT/D variant of SEQ ID NO: 13 or SEQ ID NO: 14,such as, e.g., a BoNT/D isoform or a BoNT/D subtype. In still anotheraspect of this embodiment, a BoNT/D comprises a non-naturally occurringBoNT/D variant, such as, e.g., a conservative BoNT/D variant, anon-conservative BoNT/D variant, an active BoNT/D fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D comprises a non-naturally occurring BoNT/D variant of SEQ ID NO:13 or SEQ ID NO: 14, such as, e.g., a conservative BoNT/D variant, anon-conservative BoNT/D variant, an active BoNT/D fragment, or anycombination thereof. In yet another aspect of this embodiment, a BoNT/Dcomprises a BoNT/D enzymatic domain or an active fragment thereof, aBoNT/D translocation domain or an active fragment thereof, a BoNT/Dbinding domain or an active fragment thereof, or any combinationthereof. In yet another aspect of this embodiment, a BoNT/D comprises aBoNT/D enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14, or an activefragment thereof, a BoNT/D translocation domain of SEQ ID NO: 13 or SEQID NO: 14, or an active fragment thereof, a BoNT/D binding domain of SEQID NO: 13 or SEQ ID NO: 14, or an active fragment thereof, or anycombination thereof.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 13or SEQ ID NO: 14; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to SEQ ID NO: 13 or SEQ ID NO: 14. In yetother aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,100, 200, 300, 400, or 500 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 13 or SEQ ID NO:14; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 13 or SEQ ID NO: 14. In still otheraspects of this embodiment, a BoNT/D comprises a polypeptide having,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 13 or SEQ ID NO: 14; at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 13 or SEQ ID NO: 14.

In another embodiment, a Clostridial toxin comprises a BoNT/E. In anaspect of this embodiment, a BoNT/E comprises a BoNT/E enzymatic domain,a BoNT/E translocation domain, and a BoNT/E binding domain. In anotheraspect of this embodiment, a BoNT/E comprises SEQ ID NO: 15, SEQ ID NO:16, or SEQ ID NO: 17. In another aspect of this embodiment, a BoNT/Ecomprises a naturally occurring BoNT/E variant, such as, e.g., a BoNT/Eisoform or a BoNT/E subtype. In another aspect of this embodiment, aBoNT/E comprises a naturally occurring BoNT/E variant of SEQ ID NO: 15,SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a BoNT/E isoform or aBoNT/E subtype. In still another aspect of this embodiment, a BoNT/Ecomprises a non-naturally occurring BoNT/E variant, such as, e.g., aconservative BoNT/E variant, a non-conservative BoNT/E variant, anactive BoNT/E fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/E comprises a non-naturally occurringBoNT/E variant of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, suchas, e.g., a conservative BoNT/E variant, a non-conservative BoNT/Evariant, an active BoNT/E fragment, or any combination thereof. In yetanother aspect of this embodiment, a BoNT/E comprises a BoNT/E enzymaticdomain or an active fragment thereof, a BoNT/E translocation domain oractive fragment thereof, a BoNT/E binding domain or active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/E comprises a BoNT/E enzymatic domain of SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO: 17, or active fragment thereof, aBoNT/E translocation domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ IDNO: 17, or active fragment thereof, a BoNT/E binding domain of SEQ IDNO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, or active fragment thereof, orany combination thereof.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 15,SEQ ID NO: 16, or SEQ ID NO: 17; or at most 70%, at most 75%, at most80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO: 15, SEQ IDNO: 16, or SEQ ID NO: 17. In yet other aspects of this embodiment, aBoNT/E comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; at most 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous aminoacid deletions, additions, and/or substitutions relative to SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO: 17. In still other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

In another embodiment, a Clostridial toxin comprises a BoNT/F. In anaspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domain,a BoNT/F translocation domain, and a BoNT/F binding domain. In anotheraspect of this embodiment, a BoNT/F comprises SEQ ID NO: 18, SEQ ID NO:19, or SEQ ID NO: 20. In another aspect of this embodiment, a BoNT/Fcomprises a naturally occurring BoNT/F variant, such as, e.g., a BoNT/Fisoform or a BoNT/F subtype. In another aspect of this embodiment, aBoNT/F comprises a naturally occurring BoNT/F variant of SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a BoNT/F isoform or aBoNT/F subtype. In still another aspect of this embodiment, a BoNT/Fcomprises a non-naturally occurring BoNT/F variant, such as, e.g., aconservative BoNT/F variant, a non-conservative BoNT/F variant, anactive BoNT/F fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/F comprises a non-naturally occurringBoNT/F variant of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, suchas, e.g., a conservative BoNT/F variant, a non-conservative BoNT/Fvariant, an active BoNT/F fragment, or any combination thereof. In yetanother aspect of this embodiment, a BoNT/F comprises a BoNT/F enzymaticdomain or active fragment thereof, a BoNT/F translocation domain oractive fragment thereof, a BoNT/F binding domain or active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/F comprises a BoNT/F enzymatic domain of SEQ ID NO:18, SEQ ID NO: 19, or SEQ ID NO: 20, or active fragment thereof, aBoNT/F translocation domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ IDNO: 20, or active fragment thereof, a BoNT/F binding domain of SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, or active fragment thereof, orany combination thereof.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20; or at most 70%, at most 75%, at most80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO: 18, SEQ IDNO: 19, or SEQ ID NO: 20. In yet other aspects of this embodiment, aBoNT/F comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; at most 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguous aminoacid deletions, additions, and/or substitutions relative to SEQ ID NO:18, SEQ ID NO: 19, or SEQ ID NO: 20. In still other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

In another embodiment, a Clostridial toxin comprises a BoNT/G. In anaspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domain,a BoNT/G translocation domain, and a BoNT/G binding domain. In anotheraspect of this embodiment, a BoNT/G comprises SEQ ID NO: 21. In anotheraspect of this embodiment, a BoNT/G comprises a naturally occurringBoNT/G variant, such as, e.g., a BoNT/G isoform or a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G comprises a naturallyoccurring BoNT/G variant of SEQ ID NO: 21, such as, e.g., a BoNT/Gisoform or a BoNT/G subtype of SEQ ID NO: 21. In still another aspect ofthis embodiment, a BoNT/G comprises a non-naturally occurring BoNT/Gvariant, such as, e.g., a conservative BoNT/G variant, anon-conservative BoNT/G variant or an active BoNT/G fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D comprises a non-naturally occurring BoNT/G variant of SEQ ID NO:21, such as, e.g., a conservative BoNT/G variant, a non-conservativeBoNT/G variant, an active BoNT/G fragment, or any combination thereof.In yet another aspect of this embodiment, a BoNT/G comprises a BoNT/Genzymatic domain or an active fragment thereof, a BoNT/G translocationdomain or an active fragment thereof, a BoNT/G binding domain or anactive fragment thereof, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domainof SEQ ID NO: 21 or an active fragment thereof, a BoNT/G translocationdomain of SEQ ID NO: 21 or an active fragment thereof, a BoNT/G bindingdomain of SEQ ID NO: 21 or an active fragment thereof, or anycombination thereof.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 21;or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, orat most 95% to SEQ ID NO: 21. In yet other aspects of this embodiment, aBoNT/G comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 21; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 21. In still other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 21; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 21.

In another embodiment, a Clostridial toxin comprises a TeNT. In anaspect of this embodiment, a TeNT comprises a TeNT enzymatic domain, aTeNT translocation domain, and a TeNT binding domain. In an aspect ofthis embodiment, a TeNT comprises SEQ ID NO: 22. In another aspect ofthis embodiment, a TeNT comprises a naturally occurring TeNT variant,such as, e.g., a TeNT isoform or a TeNT subtype. In another aspect ofthis embodiment, a TeNT comprises a naturally occurring TeNT variant ofSEQ ID NO: 22, such as, e.g., a TeNT isoform or a TeNT subtype. In stillanother aspect of this embodiment, a TeNT comprises a non-naturallyoccurring TeNT variant, such as, e.g., a conservative TeNT variant, anon-conservative TeNT variant, an active TeNT fragment, or anycombination thereof. In still another aspect of this embodiment, a TeNTcomprises a non-naturally occurring TeNT variant of SEQ ID NO: 22, suchas, e.g., a conservative TeNT variant, a non-conservative TeNT variant,an active TeNT fragment, or any combination thereof. In yet anotheraspect of this embodiment, a TeNT comprises a TeNT enzymatic domain oran active fragment thereof, a TeNT translocation domain or activefragment thereof, a TeNT binding domain or active fragment thereof, orany combination thereof. In yet another aspect of this embodiment, aTeNT comprises a TeNT enzymatic domain of SEQ ID NO: 22 or activefragment thereof, a TeNT translocation domain of SEQ ID NO: 22 or activefragment thereof, a TeNT binding domain of SEQ ID NO: 22 or activefragment thereof, or any combination thereof.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 22;or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, orat most 95% to SEQ ID NO: 22. In yet other aspects of this embodiment, aTeNT comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 22; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 22. In still other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 22; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 22.

In another embodiment, a Clostridial toxin comprises a BaNT. In anaspect of this embodiment, a BaNT comprises a BaNT enzymatic domain, aBaNT translocation domain, and a BaNT binding domain. In another aspectof this embodiment, a BaNT comprises SEQ ID NO: 23. In another aspect ofthis embodiment, a BaNT comprises a naturally occurring BaNT variant,such as, e.g., a BaNT isoform or a BaNT subtype. In another aspect ofthis embodiment, a BaNT comprises a naturally occurring BaNT variant ofSEQ ID NO: 23, such as, e.g., a BaNT isoform or a BaNT subtype. In stillanother aspect of this embodiment, a BaNT comprises a non-naturallyoccurring BaNT variant, such as, e.g., a conservative BaNT variant, anon-conservative BaNT variant or an active BaNT fragment, or anycombination thereof. In still another aspect of this embodiment, a BaNTcomprises a non-naturally occurring BaNT variant of SEQ ID NO: 23, suchas, e.g., a conservative BaNT variant, a non-conservative BaNT variant,an active BaNT fragment, or any combination thereof. In yet anotheraspect of this embodiment, a BaNT comprises a BaNT enzymatic domain oran active fragment thereof, a BaNT translocation domain or an activefragment thereof, a BaNT binding domain or an active fragment thereof,or any combination thereof. In yet another aspect of this embodiment, aBaNT comprises a BaNT enzymatic domain of SEQ ID NO: 23 or an activefragment thereof, a BaNT translocation domain of SEQ ID NO: 23 or anactive fragment thereof, a BaNT binding domain of SEQ ID NO: 23 or anactive fragment thereof, or any combination thereof.

In other aspects of this embodiment, a BaNT comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 23;or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, orat most 95% to SEQ ID NO: 23. In yet other aspects of this embodiment, aBaNT comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 23; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 23. In still other aspects of thisembodiment, a BaNT comprises a polypeptide having, e.g., at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 23; at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 100, 200, 300, 400, or 500 contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 23.

In another embodiment, a Clostridial toxin comprises a BuNT. In anaspect of this embodiment, a BuNT comprises a BuNT enzymatic domain, aBuNT translocation domain, and a BuNT binding domain. In another aspectof this embodiment, a BuNT comprises SEQ ID NO: 24 or SEQ ID NO: 25. Inanother aspect of this embodiment, a BuNT comprises a naturallyoccurring BuNT variant, such as, e.g., a BuNT isoform or a BuNT subtype.In another aspect of this embodiment, a BuNT comprises a naturallyoccurring BuNT variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g.,a BuNT isoform or a BuNT subtype. In still another aspect of thisembodiment, a BuNT comprises a non-naturally occurring BuNT variant,such as, e.g., a conservative BuNT variant, a non-conservative BuNTvariant, an active BuNT fragment, or any combination thereof. In stillanother aspect of this embodiment, a BuNT comprises a non-naturallyoccurring BuNT variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g.,a conservative BuNT variant, a non-conservative BuNT variant, an activeBuNT fragment, or any combination thereof. In yet another aspect of thisembodiment, a BuNT comprises a BuNT enzymatic domain or an activefragment thereof, a BuNT translocation domain or an active fragmentthereof, a BuNT binding domain, an active fragment thereof, or anycombination thereof. In yet another aspect of this embodiment, a BuNTcomprises a BuNT enzymatic domain of SEQ ID NO: 24 or SEQ ID NO: 25, oran active fragment thereof, a BuNT translocation domain of SEQ ID NO: 24or SEQ ID NO: 25, or an active fragment thereof, a BuNT binding domainof SEQ ID NO: 24 or SEQ ID NO: 25, or an active fragment thereof, or anycombination thereof.

In other aspects of this embodiment, a BuNT comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 24or SEQ ID NO: 25; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to SEQ ID NO: 24 or SEQ ID NO: 25. In yetother aspects of this embodiment, a BuNT comprises a polypeptide having,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200,300, 400, or 500 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 24 or SEQ ID NO: 25; at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500non-contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 24 or SEQ ID NO: 25. In still other aspects ofthis embodiment, a BuNT comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or500 contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 24 or SEQ ID NO: 25; at most 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, or 500 contiguous aminoacid deletions, additions, and/or substitutions relative to SEQ ID NO:24 or SEQ ID NO: 25.

As used herein, the term “Clostridial toxin chimeric” or “Clostridialtoxin chimeras” refers to a molecule comprising at least a portion fromone Clostridial toxin and a portion from at least one other protein toform a toxin with at least one property different from the referenceClostridial toxins of Table 1. Non-limiting examples of Clostridialtoxin chimeras include a Clostridial toxin comprising a non-Clostridialtoxin enzymatic domain, a Clostridial toxin comprising a non-Clostridialtoxin translocation domain, a Clostridial toxin comprising anon-Clostridial toxin binding domain, or any combination thereof. Othernon-limiting example of a Clostridial toxin chimeras include aClostridial toxin comprising a enzymatic domain from a differentClostridial toxin, a Clostridial toxin comprising a translocation domainfrom a different Clostridial toxin, a Clostridial toxin comprising abinding domain from a different Clostridial toxin, or any combinationthereof.

One class of Clostridial toxin chimeric comprises a modified Clostridialtoxin were the enzymatic domain or portion thereof, translocation domainor portion thereof, and/or binding domain or portion thereof of anaturally-occurring Clostridial toxin is either modified or replacedwith an enzymatic domain or portion thereof, translocation domain orportion thereof, and/or binding domain or portion thereof of a differentClostridial toxin. As non-limiting example, the binding domain of BoNT/Acan be replaced with the binding domain of BoNT/B producing aClostridial toxin chimeric comprising a BoNT/A enzymatic domain, aBoNT/A translocation domain, and a BoNT/B binding domain. SuchClostridial toxin chimeras are described in, e.g., J. Oliver Dolly etal., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,132,259, whichis incorporated by reference in its entirety. As another non-limitingexample, the leucine motif from BoNT/A can be inserted into the lightchain of a BoNT/E in order to increase biological persistence. SuchClostridial toxin chimeras are described in, e.g., Lance E. Steward etal., Leucine-based Motif and Clostridial Toxins, U.S. Patent Publication2003/0027752 (Feb. 6, 2003); Lance E. Steward et al., ClostridialNeurotoxin Compositions and Modified Clostridial Neurotoxins, U.S.Patent Publication 2003/0219462 (Nov. 27, 2003); and Lance E. Steward etal., Clostridial Neurotoxin Compositions and Modified ClostridialNeurotoxins, U.S. Patent Publication 2004/0220386 (Nov. 4, 2004), eachof which is incorporated by reference in its entirety.

Another class of Clostridial toxin chimeric comprises a Clostridialtoxin where the binding domain of a naturally-occurring Clostridialtoxin is either modified or replaced with a binding domain of anon-Clostridial toxin. Such Clostridial toxin chimeras possesses analtered cell binding activity because the modified toxin can either,e.g., 1) use the same receptor present on the surface of a naturallyoccurring Clostridial toxin target cell as that used by thenaturally-occurring Clostridial toxin, referred to as an enhanced cellbinding activity for a naturally-occurring Clostridial toxin targetcell; 2) use a different receptor present on the surface of a naturallyoccurring Clostridial toxin target cell, referred to as an altered cellbinding activity for a naturally-occurring Clostridial toxin targetcell; or 3) use a different receptor present on the surface of thenon-Clostridial toxin target cell, referred to as an altered cellbinding activity for a non-naturally-occurring Clostridial toxin targetcell, a re-targeted toxin or a TVEMP.

A Clostridial toxin chimeric can be a Clostridial toxin with an enhancedcell binding activity capable of intoxicating a naturally occurringClostridial toxin target cell, e.g., a motor neuron. One way thisenhanced binding activity is achieved by modifying the endogenousbinding domain of a naturally-occurring Clostridial toxin in order toenhance a cell binding activity of the toxin for its naturally-occurringreceptor. Such modifications to a targeting domain result in, e.g., aenhanced cell binding activity that increases binding affinity for anendogenous Clostridial toxin receptor present on a naturally-occurringClostridial toxin target cell; an enhanced cell binding activity thatincreases binding specificity for a subgroup of endogenous Clostridialtoxin receptors present on a naturally-occurring Clostridial toxintarget cell; or an enhanced cell binding activity that increases bothbinding affinity and binding specificity. Non-limiting examples ofmodified Clostridial toxins an enhanced cell binding activity for anaturally-occurring Clostridial toxin receptor are described in, e.g.,Lance E. Steward et al., Modified Clostridial Toxins with EnhancedTargeting Capabilities For Endogenous Clostridial Toxin ReceptorSystems, U.S. Patent Publication 2008/0096248; Lance E. Steward,Modified Clostridial Toxins with Enhanced Translocation Capabilities andEnhanced Targeting Activity for Clostridial Toxin Target Cells,International Patent Publication 2008/105901; each of which is herebyincorporated by reference in its entirety.

A Clostridial toxin chimeric can be a Clostridial toxin with an alteredcell binding activity capable of intoxicating a naturally occurringClostridial toxin target cell, e.g., a motor neuron. One way thisaltered capability is achieved by replacing the endogenous bindingdomain of a naturally-occurring Clostridial toxin with a binding domainof another molecule that preferentially binds to a different receptorpresent on the surface of a Clostridial toxin target cell. Such amodification to a binding domain results in a modified toxin that isable to preferentially bind to a non-Clostridial toxin receptor presenton a Clostridial toxin target cell. This enhanced binding activity for anaturally occurring Clostridial toxin target cell allows for lowereffective doses of a modified Clostridial toxin to be administered to anindividual because more toxin will be delivered to the target cell.Thus, modified Clostridial toxins with an enhanced binding activity willreduce the undesirable dispersal of the toxin to areas not targeted fortreatment, thereby reducing or preventing the undesirable side-effectsassociated with diffusion of a Clostridial toxin to an unwantedlocation. Non-limiting examples of modified Clostridial toxins with analtered cell binding capability for a Clostridial toxin target cell aredescribed in, e.g., Lance E. Steward et al., Multivalent ClostridialToxin Derivatives and Methods of Their Use, U.S. Pat. No. 7,514,088;Lance E. Steward et al., Modified Clostridial Toxins with AlteredTargeting Capabilities For Clostridial Toxin Target Cells, U.S. PatentPublication 2008/0161543; Lance E. Steward, Modified Clostridial Toxinswith Enhanced Translocation Capabilities and Altered Targeting Activityfor Clostridial Toxin Target Cells, U.S. Patent Publication2008/0241881; Lance E. Steward et al., Multivalent Clostridial ToxinDerivatives and Methods of Their Use, U.S. Patent Publication2009/0048431; Lance E. Steward et al., Modified Clostridial Toxins withAltered Targeting Capabilities For Clostridial Toxin Target Cells,International Patent Publication WO 2007/106115; each of which is herebyincorporated by reference in its entirety.

A Clostridial toxin chimeric can be a Clostridial toxin with an alteredcell binding activity capable of intoxicating a cell other than aClostridial toxin target cell, e.g., a cell other than a motor neuron.Called TVEMPs, these molecules achieve this intoxication by using atarget receptor present on non-Clostridial toxin target cell. Thisre-targeted capability is achieved by replacing a naturally-occurringbinding domain of a Clostridial toxin with a binding domain showing apreferential binding activity for a non-Clostridial toxin receptorpresent in a non-Clostridial toxin target cell. Such modifications to abinding domain result in a modified toxin that is able to preferentiallybind to a non-Clostridial toxin receptor present on a non-Clostridialtoxin target cell. A Clostridial toxin chimeric with an alteredtargeting activity for a non-Clostridial toxin target cell can bind to atarget receptor, translocate into the cytoplasm, and exert itsproteolytic effect on the SNARE complex of the non-Clostridial toxintarget cell. Non-limiting examples of Clostridial toxin chimeras with analtered targeting activity for a non-Clostridial toxin target cell aredescribed in, e.g., Keith A. Foster et al., Clostridial ToxinDerivatives Able To Modify Peripheral Sensory Afferent Functions, U.S.Pat. No. 5,989,545; Clifford C. Shone et al., Recombinant ToxinFragments, U.S. Pat. No. 6,461,617; Conrad P. Quinn et al., Methods andCompounds for the Treatment of Mucus Hypersecretion, U.S. Pat. No.6,632,440; Lance E. Steward et al., Methods and Compositions for theTreatment of Pancreatitis, U.S. Pat. No. 6,843,998; J. Oliver Dolly etal., Activatable Recombinant Neurotoxins, U.S. Pat. No. 7,132,259;Stephan Donovan, Clostridial Toxin Derivatives and Methods For TreatingPain, U.S. Pat. No. 7,244,437; Stephan Donovan, Clostridial ToxinDerivatives and Methods For Treating Pain, U.S. Pat. No. 7,413,742;Stephan Donovan, Clostridial Toxin Derivatives and Methods For TreatingPain, U.S. Pat. No. 7,415,338; Lance E. Steward et al., MultivalentClostridial Toxin Derivatives and Methods of Their Use, U.S. Pat. No.7,514,088; Keith A. Foster et al., Inhibition of Secretion fromNon-neural Cells, U.S. Patent Publication 2006/0216283; Keith A. Foster,Fusion Proteins, U.S. Patent Publication 2008/0064092; Keith A. Foster,Fusion Proteins, U.S. Patent Publication 2009/0035822; Lance E. Stewardet al., Multivalent Clostridial Toxin Derivatives and Methods of TheirUse, U.S. Patent Publication 2009/0048431; Keith A. Foster,Non-Cytotoxic Protein Conjugates, U.S. Patent Publication 2009/0162341;Keith A. Foster et al., Re-targeted Toxin Conjugates, InternationalPatent Publication WO 2005/023309; and Lance E. Steward, ModifiedClostridial Toxins with Enhanced Translocation Capabilities and AlteredTargeting Capabilities for Non-Clostridial Toxin Target Cells,International Patent Application WO 2008/008805; each of which is herebyincorporated by reference in its entirety.

Aspects of the present specification provide, in part, a Clostridialtoxin enzymatic domain. As used herein, the term “Clostridial toxinenzymatic domain” refers to any Clostridial toxin polypeptide that canexecute the enzymatic target modification step of the intoxicationprocess. Thus, a Clostridial toxin enzymatic domain specifically targetsa Clostridial toxin substrate and encompasses the proteolytic cleavageof a Clostridial toxin substrate, such as, e.g., SNARE proteins like aSNAP-25 substrate, a VAMP substrate, and a Syntaxin substrate.Non-limiting examples of a Clostridial toxin enzymatic domain include,e.g., a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a BoNT/C1enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E enzymatic domain,a BoNT/F enzymatic domain, a BoNT/G enzymatic domain, a TeNT enzymaticdomain, a BaNT enzymatic domain, and a BuNT enzymatic domain.

A Clostridial toxin enzymatic domain includes, without limitation,naturally occurring Clostridial toxin enzymatic domain variants, suchas, e.g., Clostridial toxin enzymatic domain isoforms and Clostridialtoxin enzymatic domain subtypes; and non-naturally occurring Clostridialtoxin enzymatic domain variants, such as, e.g., conservative Clostridialtoxin enzymatic domain variants, non-conservative Clostridial toxinenzymatic domain variants, active Clostridial toxin enzymatic domainfragments thereof, or any combination thereof.

As used herein, the term “Clostridial toxin enzymatic domain variant,”whether naturally-occurring or non-naturally-occurring, refers to aClostridial toxin enzymatic domain that has at least one amino acidchange from the corresponding region of the disclosed referencesequences (Table 1) and can be described in percent identity to thecorresponding region of that reference sequence. Unless expresslyindicated, Clostridial toxin enzymatic domain variants useful topractice disclosed embodiments are variants that execute the enzymatictarget modification step of the intoxication process. As non-limitingexamples, a BoNT/A enzymatic domain variant will have at least one aminoacid difference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 1/2-429 of SEQ ID NO: 1; a BoNT/Benzymatic domain variant will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to amino acids 1/2-436 of SEQ ID NO: 6; a BoNT/C1 enzymaticdomain variant will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared toamino acids 1/2-436 of SEQ ID NO: 11; a BoNT/D enzymatic domain variantwill have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to amino acids1/2-436 of SEQ ID NO: 13; a BoNT/E enzymatic domain variant will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to amino acids 1/2-411of SEQ ID NO: 15; a BoNT/F enzymatic domain variant will have at leastone amino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to amino acids 1/2-428 of SEQ ID NO:18; a BoNT/G enzymatic domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 1/2-438 of SEQ ID NO: 21; a TeNTenzymatic domain variant will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to amino acids 1/2-438 of SEQ ID NO: 22; a BaNT enzymaticdomain variant will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared toamino acids 1/2-420 of SEQ ID NO: 23; and a BuNT enzymatic domainvariant will have at least one amino acid difference, such as, e.g., anamino acid substitution, deletion or addition, as compared to aminoacids 1/2-411 of SEQ ID NO: 24.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinenzymatic domain variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3, BoNT/A4, and BoNT/A5, with specific enzymatic domain subtypesshowing about 80% to 95% amino acid identity when compared to the BoNT/Aenzymatic domain of SEQ ID NO: 1. As used herein, the term “naturallyoccurring Clostridial toxin enzymatic domain variant” refers to anyClostridial toxin enzymatic domain produced by a naturally-occurringprocess, including, without limitation, Clostridial toxin enzymaticdomain isoforms produced from alternatively-spliced transcripts,Clostridial toxin enzymatic domain isoforms produced by spontaneousmutation and Clostridial toxin enzymatic domain subtypes. A naturallyoccurring Clostridial toxin enzymatic domain variant can function insubstantially the same manner as the reference Clostridial toxinenzymatic domain on which the naturally occurring Clostridial toxinenzymatic domain variant is based, and can be substituted for thereference Clostridial toxin enzymatic domain in any aspect of thepresent specification.

A non-limiting examples of a naturally occurring Clostridial toxinenzymatic domain variant is a Clostridial toxin enzymatic domain isoformsuch as, e.g., a BoNT/A enzymatic domain isoform, a BoNT/B enzymaticdomain isoform, a BoNT/C1 enzymatic domain isoform, a BoNT/D enzymaticdomain isoform, a BoNT/E enzymatic domain isoform, a BoNT/F enzymaticdomain isoform, a BoNT/G enzymatic domain isoform, a TeNT enzymaticdomain isoform, a BaNT enzymatic domain isoform, and a BuNT enzymaticdomain isoform. Another non-limiting examples of a naturally occurringClostridial toxin enzymatic domain variant is a Clostridial toxinenzymatic domain subtype such as, e.g., an enzymatic domain from subtypeBoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, or BoNT/A5; an enzymatic domain fromsubtype BoNT/B1, BoNT/B2, BoNT/Bbv, or BoNT/Bnp; an enzymatic domainfrom subtype BoNT/C1-1 or BoNT/C1-2; an enzymatic domain from subtypeBoNT/E1, BoNT/E2 and BoNT/E3; an enzymatic domain from subtype BoNT/F1,BoNT/F2, or BoNT/F3; and an enzymatic domain from subtype BuNT-1 orBuNT-2.

As used herein, the term “non-naturally occurring Clostridial toxinenzymatic domain variant” refers to any Clostridial toxin enzymaticdomain produced with the aid of human manipulation, including, withoutlimitation, Clostridial toxin enzymatic domains produced by geneticengineering using random mutagenesis or rational design and Clostridialtoxin enzymatic domains produced by chemical synthesis. Non-limitingexamples of non-naturally occurring Clostridial toxin enzymatic domainvariants include, e.g., conservative Clostridial toxin enzymatic domainvariants, non-conservative Clostridial toxin enzymatic domain variants,Clostridial toxin enzymatic domain chimeric variants, and activeClostridial toxin enzymatic domain fragments.

As used herein, the term “conservative Clostridial toxin enzymaticdomain variant” refers to a Clostridial toxin enzymatic domain that hasat least one amino acid substituted by another amino acid or an aminoacid analog that has at least one property similar to that of theoriginal amino acid from the reference Clostridial toxin enzymaticdomain sequence (Table 1). Examples of properties include, withoutlimitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative Clostridial toxin enzymaticdomain variant can function in substantially the same manner as thereference Clostridial toxin enzymatic domain on which the conservativeClostridial toxin enzymatic domain variant is based, and can besubstituted for the reference Clostridial toxin enzymatic domain in anyaspect of the present specification. Non-limiting examples of aconservative Clostridial toxin enzymatic domain variant include, e.g.,conservative BoNT/A enzymatic domain variants, conservative BoNT/Benzymatic domain variants, conservative BoNT/C1 enzymatic domainvariants, conservative BoNT/D enzymatic domain variants, conservativeBoNT/E enzymatic domain variants, conservative BoNT/F enzymatic domainvariants, conservative BoNT/G enzymatic domain variants, conservativeTeNT enzymatic domain variants, conservative BaNT enzymatic domainvariants, and conservative BuNT enzymatic domain variants.

As used herein, the term “non-conservative Clostridial toxin enzymaticdomain variant” refers to a Clostridial toxin enzymatic domain inwhich 1) at least one amino acid is deleted from the referenceClostridial toxin enzymatic domain on which the non-conservativeClostridial toxin enzymatic domain variant is based; 2) at least oneamino acid added to the reference Clostridial toxin enzymatic domain onwhich the non-conservative Clostridial toxin enzymatic domain is based;or 3) at least one amino acid is substituted by another amino acid or anamino acid analog that does not share any property similar to that ofthe original amino acid from the reference Clostridial toxin enzymaticdomain sequence (Table 1). A non-conservative Clostridial toxinenzymatic domain variant can function in substantially the same manneras the reference Clostridial toxin enzymatic domain on which thenon-conservative Clostridial toxin enzymatic domain variant is based,and can be substituted for the reference Clostridial toxin enzymaticdomain in any aspect of the present specification. Non-limiting examplesof a non-conservative Clostridial toxin enzymatic domain variantinclude, e.g., non-conservative BoNT/A enzymatic domain variants,non-conservative BoNT/B enzymatic domain variants, non-conservativeBoNT/C1 enzymatic domain variants, non-conservative BoNT/D enzymaticdomain variants, non-conservative BoNT/E enzymatic domain variants,non-conservative BoNT/F enzymatic domain variants, non-conservativeBoNT/G enzymatic domain variants, and non-conservative TeNT enzymaticdomain variants, non-conservative BaNT enzymatic domain variants, andnon-conservative BuNT enzymatic domain variants.

As used herein, the term “active Clostridial toxin enzymatic domainfragment” refers to any of a variety of Clostridial toxin fragmentscomprising the enzymatic domain can be useful in aspects of the presentspecification with the proviso that these enzymatic domain fragments canspecifically target the core components of the neurotransmitter releaseapparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The enzymatic domains of Clostridial toxins are approximately420-460 amino acids in length and comprise an enzymatic domain (Table1). Research has shown that the entire length of a Clostridial toxinenzymatic domain is not necessary for the enzymatic activity of theenzymatic domain. As a non-limiting example, the first eight amino acidsof the BoNT/A enzymatic domain are not required for enzymatic activity.As another non-limiting example, the first eight amino acids of the TeNTenzymatic domain are not required for enzymatic activity. Likewise, thecarboxyl-terminus of the enzymatic domain is not necessary for activity.As a non-limiting example, the last 32 amino acids of the BoNT/Aenzymatic domain are not required for enzymatic activity. As anothernon-limiting example, the last 31 amino acids of the TeNT enzymaticdomain are not required for enzymatic activity. Thus, aspects of thisembodiment include Clostridial toxin enzymatic domains comprising anenzymatic domain having a length of, e.g., at least 350, 375, 400, 425,or 450 amino acids. Other aspects of this embodiment include Clostridialtoxin enzymatic domains comprising an enzymatic domain having a lengthof, e.g., at most 350, 375, 400, 425, or 450 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin enzymaticdomain variants and non-naturally-occurring Clostridial toxin enzymaticdomain variants, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin enzymatic domain. Inan aspect of this embodiment, a Clostridial toxin enzymatic domaincomprises a naturally occurring Clostridial toxin enzymatic domainvariant, such as, e.g., a Clostridial toxin enzymatic domain isoform ora Clostridial toxin enzymatic domain subtype. In another aspect of thisembodiment, a Clostridial toxin enzymatic domain comprises anon-naturally occurring Clostridial toxin enzymatic domain variant, suchas, e.g., a conservative Clostridial toxin enzymatic domain variant, anon-conservative Clostridial toxin enzymatic domain variant, an activeClostridial toxin enzymatic domain fragment, or any combination thereof.

In another embodiment, a hydrophobic amino acid at one particularposition in the polypeptide chain of the Clostridial toxin enzymaticdomain can be substituted with another hydrophobic amino acid. Examplesof hydrophobic amino acids include, e.g., C, F, I, L, M, V and W. Inanother aspect of this embodiment, an aliphatic amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinenzymatic domain can be substituted with another aliphatic amino acid.Examples of aliphatic amino acids include, e.g., A, I, L, P, and V. Inyet another aspect of this embodiment, an aromatic amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinenzymatic domain can be substituted with another aromatic amino acid.Examples of aromatic amino acids include, e.g., F, H, W and Y. In stillanother aspect of this embodiment, a stacking amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinenzymatic domain can be substituted with another stacking amino acid.Examples of stacking amino acids include, e.g., F, H, W and Y. In afurther aspect of this embodiment, a polar amino acid at one particularposition in the polypeptide chain of the Clostridial toxin enzymaticdomain can be substituted with another polar amino acid. Examples ofpolar amino acids include, e.g., D, E, K, N, Q, and R. In a furtheraspect of this embodiment, a less polar or indifferent amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinenzymatic domain can be substituted with another less polar orindifferent amino acid. Examples of less polar or indifferent aminoacids include, e.g., A, H, G, P, S, T, and Y. In a yet further aspect ofthis embodiment, a positive charged amino acid at one particularposition in the polypeptide chain of the Clostridial toxin enzymaticdomain can be substituted with another positive charged amino acid.Examples of positive charged amino acids include, e.g., K, R, and H. Ina still further aspect of this embodiment, a negative charged amino acidat one particular position in the polypeptide chain of the Clostridialtoxin enzymatic domain can be substituted with another negative chargedamino acid. Examples of negative charged amino acids include, e.g., Dand E. In another aspect of this embodiment, a small amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinenzymatic domain can be substituted with another small amino acid.Examples of small amino acids include, e.g., A, D, G, N, P, S, and T. Inyet another aspect of this embodiment, a C-beta branching amino acid atone particular position in the polypeptide chain of the Clostridialtoxin enzymatic domain can be substituted with another C-beta branchingamino acid. Examples of C-beta branching amino acids include, e.g., I, Tand V.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/A enzymatic domain. In an aspect of this embodiment, a BoNT/Aenzymatic domain comprises the enzymatic domains of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In other aspects ofthis embodiment, a BoNT/A enzymatic domain comprises amino acids 1/2-429of SEQ ID NO: 1. In another aspect of this embodiment, a BoNT/Aenzymatic domain comprises a naturally occurring BoNT/A enzymatic domainvariant, such as, e.g., an enzymatic domain from a BoNT/A isoform or anenzymatic domain from a BoNT/A subtype. In another aspect of thisembodiment, a BoNT/A enzymatic domain comprises a naturally occurringBoNT/A enzymatic domain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a BoNT/A isoformenzymatic domain or a BoNT/A subtype enzymatic domain. In another aspectof this embodiment, a BoNT/A enzymatic domain comprises amino acids1/2-429 of a naturally occurring BoNT/A enzymatic domain variant of SEQID NO: 1, such as, e.g., a BoNT/A isoform enzymatic domain or a BoNT/Asubtype enzymatic domain. In still another aspect of this embodiment, aBoNT/A enzymatic domain comprises a non-naturally occurring BoNT/Aenzymatic domain variant, such as, e.g., a conservative BoNT/A enzymaticdomain variant, a non-conservative BoNT/A enzymatic domain variant, anactive BoNT/A enzymatic domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BoNT/A enzymatic domaincomprises the enzymatic domain of a non-naturally occurring BoNT/Aenzymatic domain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a conservative BoNT/Aenzymatic domain variant, a non-conservative BoNT/A enzymatic domainvariant, an active BoNT/A enzymatic domain fragment, or any combinationthereof. In still another aspect of this embodiment, a BoNT/A enzymaticdomain comprises amino acids 1/2-429 of a non-naturally occurring BoNT/Aenzymatic domain variant of SEQ ID NO: 1, such as, e.g., a conservativeBoNT/A enzymatic domain variant, a non-conservative BoNT/A enzymaticdomain variant, an active BoNT/A enzymatic domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BoNT/A enzymatic domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5; or at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, or at most 95% to the enzymatic domain of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/A enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 1/2-429 of SEQ ID NO: 1; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to amino acids1/2-429 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous aminoacid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/Aenzymatic domain comprises a polypeptide having, e.g., at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-429 of SEQ ID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to amino acids 1/2-429 of SEQ ID NO: 1. Instill other aspects of this embodiment, a BoNT/A enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO: 5. In further other aspects of this embodiment, aBoNT/A enzymatic domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to amino acids1/2-429 of SEQ ID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 1/2-429 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/B enzymatic domain. In an aspect of this embodiment, a BoNT/Benzymatic domain comprises the enzymatic domains of SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In other aspects ofthis embodiment, a BoNT/B enzymatic domain comprises amino acids 1/2-436of SEQ ID NO: 6. In another aspect of this embodiment, a BoNT/Benzymatic domain comprises a naturally occurring BoNT/B enzymatic domainvariant, such as, e.g., an enzymatic domain from a BoNT/B isoform or anenzymatic domain from a BoNT/B subtype. In another aspect of thisembodiment, a BoNT/B enzymatic domain comprises a naturally occurringBoNT/B enzymatic domain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a BoNT/B isoformenzymatic domain or a BoNT/B subtype enzymatic domain. In another aspectof this embodiment, a BoNT/B enzymatic domain comprises amino acids1/2-436 of a naturally occurring BoNT/B enzymatic domain variant of SEQID NO: 6, such as, e.g., a BoNT/B isoform enzymatic domain or a BoNT/Bsubtype enzymatic domain. In still another aspect of this embodiment, aBoNT/B enzymatic domain comprises a non-naturally occurring BoNT/Benzymatic domain variant, such as, e.g., a conservative BoNT/B enzymaticdomain variant, a non-conservative BoNT/B enzymatic domain variant, anactive BoNT/B enzymatic domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BoNT/B enzymatic domaincomprises the enzymatic domain of a non-naturally occurring BoNT/Benzymatic domain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a conservative BoNT/Benzymatic domain variant, a non-conservative BoNT/B enzymatic domainvariant, an active BoNT/B enzymatic domain fragment, or any combinationthereof. In still another aspect of this embodiment, a BoNT/B enzymaticdomain comprises amino acids 1/2-436 of a non-naturally occurring BoNT/Benzymatic domain variant of SEQ ID NO: 6, such as, e.g., a conservativeBoNT/B enzymatic domain variant, a non-conservative BoNT/B enzymaticdomain variant, an active BoNT/B enzymatic domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BoNT/B enzymatic domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or SEQ ID NO: 10; or at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, or at most 95% to the enzymatic domain of SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Inyet other aspects of this embodiment, a BoNT/B enzymatic domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to amino acids 1/2-436 of SEQ ID NO: 6; or at most 70%, at most 75%,at most 80%, at most 85%, at most 90%, or at most 95% to amino acids1/2-436 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/B enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; or at most1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguousamino acid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10. In yet other aspects of this embodiment, a BoNT/Benzymatic domain comprises a polypeptide having, e.g., at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-436 of SEQ ID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to amino acids 1/2-436 of SEQ ID NO: 6. Instill other aspects of this embodiment, a BoNT/B enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10. In further other aspects of this embodiment, aBoNT/B enzymatic domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to amino acids1/2-436 of SEQ ID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 1/2-436 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/C1 enzymatic domain. In an aspect of this embodiment, a BoNT/C1enzymatic domain comprises the enzymatic domains of SEQ ID NO: 11 or SEQID NO: 12. In other aspects of this embodiment, a BoNT/C1 enzymaticdomain comprises amino acids 1/2-436 of SEQ ID NO: 11. In another aspectof this embodiment, a BoNT/C1 enzymatic domain comprises a naturallyoccurring BoNT/C1 enzymatic domain variant, such as, e.g., an enzymaticdomain from a BoNT/C1 isoform or an enzymatic domain from a BoNT/C1subtype. In another aspect of this embodiment, a BoNT/C1 enzymaticdomain comprises a naturally occurring BoNT/C1 enzymatic domain variantof SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., a BoNT/C1 isoformenzymatic domain or a BoNT/C1 subtype enzymatic domain. In anotheraspect of this embodiment, a BoNT/C1 enzymatic domain comprises aminoacids 1/2-436 of a naturally occurring BoNT/C1 enzymatic domain variantof SEQ ID NO: 11, such as, e.g., a BoNT/C1 isoform enzymatic domain or aBoNT/C1 subtype enzymatic domain. In still another aspect of thisembodiment, a BoNT/C1 enzymatic domain comprises a non-naturallyoccurring BoNT/C1 enzymatic domain variant, such as, e.g., aconservative BoNT/C1 enzymatic domain variant, a non-conservativeBoNT/C1 enzymatic domain variant, an active BoNT/C1 enzymatic domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/C1 enzymatic domain comprises the enzymatic domain ofa non-naturally occurring BoNT/C1 enzymatic domain variant of SEQ ID NO:11 or SEQ ID NO: 12, such as, e.g., a conservative BoNT/C1 enzymaticdomain variant, a non-conservative BoNT/C1 enzymatic domain variant, anactive BoNT/C1 enzymatic domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BoNT/C1 enzymatic domaincomprises amino acids 1/2-436 of a non-naturally occurring BoNT/C1enzymatic domain variant of SEQ ID NO: 11, such as, e.g., a conservativeBoNT/C1 enzymatic domain variant, a non-conservative BoNT/C1 enzymaticdomain variant, an active BoNT/C1 enzymatic domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to the enzymatic domain of SEQ ID NO: 11 or SEQ ID NO: 12. In yetother aspects of this embodiment, a BoNT/C1 enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 1/2-436 of SEQ ID NO: 11; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to amino acids1/2-436 of SEQ ID NO: 11.

In other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 11 or SEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 11or SEQ ID NO: 12. In yet other aspects of this embodiment, a BoNT/C1enzymatic domain comprises a polypeptide having, e.g., at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-436 of SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to amino acids 1/2-436 of SEQ ID NO: 11.In still other aspects of this embodiment, a BoNT/C1 enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 11 or SEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 11or SEQ ID NO: 12. In further other aspects of this embodiment, a BoNT/C1enzymatic domain comprises a polypeptide having, e.g., at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-436 of SEQ ID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 1/2-436 of SEQ ID NO: 11.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/D enzymatic domain. In an aspect of this embodiment, a BoNT/Denzymatic domain comprises the enzymatic domains of SEQ ID NO: 13 or SEQID NO: 14. In other aspects of this embodiment, a BoNT/D enzymaticdomain comprises amino acids 1/2-436 of SEQ ID NO: 13. In another aspectof this embodiment, a BoNT/D enzymatic domain comprises a naturallyoccurring BoNT/D enzymatic domain variant, such as, e.g., an enzymaticdomain from a BoNT/D isoform or an enzymatic domain from a BoNT/Dsubtype. In another aspect of this embodiment, a BoNT/D enzymatic domaincomprises a naturally occurring BoNT/D enzymatic domain variant of SEQID NO: 13 or SEQ ID NO: 14, such as, e.g., a BoNT/D isoform enzymaticdomain or a BoNT/D subtype enzymatic domain. In another aspect of thisembodiment, a BoNT/D enzymatic domain comprises amino acids 1/2-436 of anaturally occurring BoNT/D enzymatic domain variant of SEQ ID NO: 13,such as, e.g., a BoNT/D isoform enzymatic domain or a BoNT/D subtypeenzymatic domain. In still another aspect of this embodiment, a BoNT/Denzymatic domain comprises a non-naturally occurring BoNT/D enzymaticdomain variant, such as, e.g., a conservative BoNT/D enzymatic domainvariant, a non-conservative BoNT/D enzymatic domain variant, an activeBoNT/D enzymatic domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/D enzymatic domain comprisesthe enzymatic domain of a non-naturally occurring BoNT/D enzymaticdomain variant of SEQ ID NO: 13 or SEQ ID NO: 14, such as, e.g., aconservative BoNT/D enzymatic domain variant, a non-conservative BoNT/Denzymatic domain variant, an active BoNT/D enzymatic domain fragment, orany combination thereof. In still another aspect of this embodiment, aBoNT/D enzymatic domain comprises amino acids 1/2-436 of a non-naturallyoccurring BoNT/D enzymatic domain variant of SEQ ID NO: 13, such as,e.g., a conservative BoNT/D enzymatic domain variant, a non-conservativeBoNT/D enzymatic domain variant, an active BoNT/D enzymatic domainfragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/D enzymatic domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most 70%,at most 75%, at most 80%, at most 85%, at most 90%, or at most 95% tothe enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14. In yet otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 1/2-436 of SEQ ID NO: 13; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to amino acids1/2-436 of SEQ ID NO: 13.

In other aspects of this embodiment, a BoNT/D enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 13or SEQ ID NO: 14; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the enzymatic domain of SEQ ID NO: 13 or SEQID NO: 14. In yet other aspects of this embodiment, a BoNT/D enzymaticdomain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-436 of SEQID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-436 of SEQ ID NO: 13. In still other aspectsof this embodiment, a BoNT/D enzymatic domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to the enzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14; orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 13 or SEQ ID NO: 14. In further otheraspects of this embodiment, a BoNT/D enzymatic domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 1/2-436 of SEQ ID NO: 13; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 1/2-436 of SEQ ID NO: 13.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/E enzymatic domain. In an aspect of this embodiment, a BoNT/Eenzymatic domain comprises the enzymatic domains of SEQ ID NO: 15, SEQID NO: 16, or SEQ ID NO: 17. In other aspects of this embodiment, aBoNT/E enzymatic domain comprises amino acids 1/2-411 of SEQ ID NO: 15.In another aspect of this embodiment, a BoNT/E enzymatic domaincomprises a naturally occurring BoNT/E enzymatic domain variant, suchas, e.g., an enzymatic domain from a BoNT/E isoform or an enzymaticdomain from a BoNT/E subtype. In another aspect of this embodiment, aBoNT/E enzymatic domain comprises a naturally occurring BoNT/E enzymaticdomain variant of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, suchas, e.g., a BoNT/E isoform enzymatic domain or a BoNT/E subtypeenzymatic domain. In another aspect of this embodiment, a BoNT/Eenzymatic domain comprises amino acids 1/2-411 of a naturally occurringBoNT/E enzymatic domain variant of SEQ ID NO: 15, such as, e.g., aBoNT/E isoform enzymatic domain or a BoNT/E subtype enzymatic domain. Instill another aspect of this embodiment, a BoNT/E enzymatic domaincomprises a non-naturally occurring BoNT/E enzymatic domain variant,such as, e.g., a conservative BoNT/E enzymatic domain variant, anon-conservative BoNT/E enzymatic domain variant, an active BoNT/Eenzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/E enzymatic domain comprises theenzymatic domain of a non-naturally occurring BoNT/E enzymatic domainvariant of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, such as,e.g., a conservative BoNT/E enzymatic domain variant, a non-conservativeBoNT/E enzymatic domain variant, an active BoNT/E enzymatic domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/E enzymatic domain comprises amino acids 1/2-411 of anon-naturally occurring BoNT/E enzymatic domain variant of SEQ ID NO:15, such as, e.g., a conservative BoNT/E enzymatic domain variant, anon-conservative BoNT/E enzymatic domain variant, an active BoNT/Eenzymatic domain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/E enzymatic domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17;or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, orat most 95% to the enzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17. In yet other aspects of this embodiment, a BoNT/Eenzymatic domain comprises a polypeptide having an amino acid identityof, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% to amino acids 1/2-411 of SEQ ID NO: 15; orat most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or atmost 95% to amino acids 1/2-411 of SEQ ID NO: 15.

In other aspects of this embodiment, a BoNT/E enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 15,SEQ ID NO: 16, or SEQ ID NO: 17; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In yet other aspects of thisembodiment, a BoNT/E enzymatic domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-411 of SEQ ID NO: 15; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-411 of SEQ ID NO: 15. In still other aspects of this embodiment, aBoNT/E enzymatic domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. Infurther other aspects of this embodiment, a BoNT/E enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-411 of SEQID NO: 15; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-411 of SEQ ID NO: 15.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/F enzymatic domain. In an aspect of this embodiment, a BoNT/Fenzymatic domain comprises the enzymatic domains of SEQ ID NO: 18, SEQID NO: 19, or SEQ ID NO: 20. In other aspects of this embodiment, aBoNT/F enzymatic domain comprises amino acids 1/2-428 of SEQ ID NO: 18.In another aspect of this embodiment, a BoNT/F enzymatic domaincomprises a naturally occurring BoNT/F enzymatic domain variant, suchas, e.g., an enzymatic domain from a BoNT/F isoform or an enzymaticdomain from a BoNT/F subtype. In another aspect of this embodiment, aBoNT/F enzymatic domain comprises a naturally occurring BoNT/F enzymaticdomain variant of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, suchas, e.g., a BoNT/F isoform enzymatic domain or a BoNT/F subtypeenzymatic domain. In another aspect of this embodiment, a BoNT/Fenzymatic domain comprises amino acids 1/2-428 of a naturally occurringBoNT/F enzymatic domain variant of SEQ ID NO: 18, such as, e.g., aBoNT/F isoform enzymatic domain or a BoNT/F subtype enzymatic domain. Instill another aspect of this embodiment, a BoNT/F enzymatic domaincomprises a non-naturally occurring BoNT/F enzymatic domain variant,such as, e.g., a conservative BoNT/F enzymatic domain variant, anon-conservative BoNT/F enzymatic domain variant, an active BoNT/Fenzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/F enzymatic domain comprises theenzymatic domain of a non-naturally occurring BoNT/F enzymatic domainvariant of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, such as,e.g., a conservative BoNT/F enzymatic domain variant, a non-conservativeBoNT/F enzymatic domain variant, an active BoNT/F enzymatic domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/F enzymatic domain comprises amino acids 1/2-428 of anon-naturally occurring BoNT/F enzymatic domain variant of SEQ ID NO:18, such as, e.g., a conservative BoNT/F enzymatic domain variant, anon-conservative BoNT/F enzymatic domain variant, an active BoNT/Fenzymatic domain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/F enzymatic domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20;or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, orat most 95% to the enzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, orSEQ ID NO: 20. In yet other aspects of this embodiment, a BoNT/Fenzymatic domain comprises a polypeptide having an amino acid identityof, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% to amino acids 1/2-428 of SEQ ID NO: 18; orat most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or atmost 95% to amino acids 1/2-428 of SEQ ID NO: 18.

In other aspects of this embodiment, a BoNT/F enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In yet other aspects of thisembodiment, a BoNT/F enzymatic domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-428 of SEQ ID NO: 18; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-428 of SEQ ID NO: 18. In still other aspects of this embodiment, aBoNT/F enzymatic domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. Infurther other aspects of this embodiment, a BoNT/F enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-428 of SEQID NO: 18; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-428 of SEQ ID NO: 18.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBoNT/G enzymatic domain. In an aspect of this embodiment, a BoNT/Genzymatic domain comprises the enzymatic domains of SEQ ID NO: 21. Inother aspects of this embodiment, a BoNT/G enzymatic domain comprisesamino acids 1/2-4435 of SEQ ID NO: 21. In another aspect of thisembodiment, a BoNT/G enzymatic domain comprises a naturally occurringBoNT/G enzymatic domain variant, such as, e.g., an enzymatic domain froma BoNT/G isoform or an enzymatic domain from a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G enzymatic domain comprises anaturally occurring BoNT/G enzymatic domain variant of SEQ ID NO: 21,such as, e.g., a BoNT/G isoform enzymatic domain or a BoNT/G subtypeenzymatic domain. In another aspect of this embodiment, a BoNT/Genzymatic domain comprises amino acids 1/2-4435 of a naturally occurringBoNT/G enzymatic domain variant of SEQ ID NO: 21, such as, e.g., aBoNT/G isoform enzymatic domain or a BoNT/G subtype enzymatic domain. Instill another aspect of this embodiment, a BoNT/G enzymatic domaincomprises a non-naturally occurring BoNT/G enzymatic domain variant,such as, e.g., a conservative BoNT/G enzymatic domain variant, anon-conservative BoNT/G enzymatic domain variant, an active BoNT/Genzymatic domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BoNT/G enzymatic domain comprises theenzymatic domain of a non-naturally occurring BoNT/G enzymatic domainvariant of SEQ ID NO: 21, such as, e.g., a conservative BoNT/G enzymaticdomain variant, a non-conservative BoNT/G enzymatic domain variant, anactive BoNT/G enzymatic domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BoNT/G enzymatic domaincomprises amino acids 1/2-4435 of a non-naturally occurring BoNT/Genzymatic domain variant of SEQ ID NO: 21, such as, e.g., a conservativeBoNT/G enzymatic domain variant, a non-conservative BoNT/G enzymaticdomain variant, an active BoNT/G enzymatic domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BoNT/G enzymatic domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 21; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the enzymaticdomain of SEQ ID NO: 21. In yet other aspects of this embodiment, aBoNT/G enzymatic domain comprises a polypeptide having an amino acididentity of, e.g., at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% to amino acids 1/2-4435 of SEQ ID NO:21; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,or at most 95% to amino acids 1/2-4435 of SEQ ID NO: 21.

In other aspects of this embodiment, a BoNT/G enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 21;or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the enzymatic domain of SEQ ID NO: 21. In yet other aspectsof this embodiment, a BoNT/G enzymatic domain comprises a polypeptidehaving, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-4435 of SEQ ID NO: 21; or at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-4435 of SEQ ID NO: 21. In still other aspects of this embodiment, aBoNT/G enzymatic domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to theenzymatic domain of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 21.In further other aspects of this embodiment, a BoNT/G enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-4435 of SEQID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-4435 of SEQ ID NO: 21.

In another embodiment, a Clostridial toxin enzymatic domain comprises aTeNT enzymatic domain. In an aspect of this embodiment, a TeNT enzymaticdomain comprises the enzymatic domains of SEQ ID NO: 22. In otheraspects of this embodiment, a TeNT enzymatic domain comprises aminoacids 1/2-438 of SEQ ID NO: 22. In another aspect of this embodiment, aTeNT enzymatic domain comprises a naturally occurring TeNT enzymaticdomain variant, such as, e.g., an enzymatic domain from a TeNT isoformor an enzymatic domain from a TeNT subtype. In another aspect of thisembodiment, a TeNT enzymatic domain comprises a naturally occurring TeNTenzymatic domain variant of SEQ ID NO: 22, such as, e.g., a TeNT isoformenzymatic domain or a TeNT subtype enzymatic domain. In another aspectof this embodiment, a TeNT enzymatic domain comprises amino acids1/2-438 of a naturally occurring TeNT enzymatic domain variant of SEQ IDNO: 22, such as, e.g., a TeNT isoform enzymatic domain or a TeNT subtypeenzymatic domain. In still another aspect of this embodiment, a TeNTenzymatic domain comprises a non-naturally occurring TeNT enzymaticdomain variant, such as, e.g., a conservative TeNT enzymatic domainvariant, a non-conservative TeNT enzymatic domain variant, an activeTeNT enzymatic domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a TeNT enzymatic domain comprises theenzymatic domain of a non-naturally occurring TeNT enzymatic domainvariant of SEQ ID NO: 22, such as, e.g., a conservative TeNT enzymaticdomain variant, a non-conservative TeNT enzymatic domain variant, anactive TeNT enzymatic domain fragment, or any combination thereof. Instill another aspect of this embodiment, a TeNT enzymatic domaincomprises amino acids 1/2-438 of a non-naturally occurring TeNTenzymatic domain variant of SEQ ID NO: 22, such as, e.g., a conservativeTeNT enzymatic domain variant, a non-conservative TeNT enzymatic domainvariant, an active TeNT enzymatic domain fragment, or any combinationthereof.

In other aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 22; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the enzymaticdomain of SEQ ID NO: 22. In yet other aspects of this embodiment, a TeNTenzymatic domain comprises a polypeptide having an amino acid identityof, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% to amino acids 1/2-438 of SEQ ID NO: 22; orat most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or atmost 95% to amino acids 1/2-438 of SEQ ID NO: 22.

In other aspects of this embodiment, a TeNT enzymatic domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the enzymatic domain of SEQ ID NO: 22; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the enzymatic domain of SEQ ID NO: 22. In yet other aspectsof this embodiment, a TeNT enzymatic domain comprises a polypeptidehaving, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-438 of SEQ ID NO: 22; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-438 of SEQ ID NO: 22. In still other aspects of this embodiment, aTeNT enzymatic domain comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the enzymaticdomain of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the enzymatic domain of SEQ ID NO: 22. Infurther other aspects of this embodiment, a TeNT enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-438 of SEQID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-438 of SEQ ID NO: 22.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBaNT enzymatic domain. In an aspect of this embodiment, a BaNT enzymaticdomain comprises the enzymatic domains of SEQ ID NO: 23. In otheraspects of this embodiment, a BaNT enzymatic domain comprises aminoacids 1/2-420 of SEQ ID NO: 23. In another aspect of this embodiment, aBaNT enzymatic domain comprises a naturally occurring BaNT enzymaticdomain variant, such as, e.g., an enzymatic domain from a BaNT isoformor an enzymatic domain from a BaNT subtype. In another aspect of thisembodiment, a BaNT enzymatic domain comprises a naturally occurring BaNTenzymatic domain variant of SEQ ID NO: 23, such as, e.g., a BaNT isoformenzymatic domain or a BaNT subtype enzymatic domain. In another aspectof this embodiment, a BaNT enzymatic domain comprises amino acids1/2-420 of a naturally occurring BaNT enzymatic domain variant of SEQ IDNO: 23, such as, e.g., a BaNT isoform enzymatic domain or a BaNT subtypeenzymatic domain. In still another aspect of this embodiment, a BaNTenzymatic domain comprises a non-naturally occurring BaNT enzymaticdomain variant, such as, e.g., a conservative BaNT enzymatic domainvariant, a non-conservative BaNT enzymatic domain variant, an activeBaNT enzymatic domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BaNT enzymatic domain comprises theenzymatic domain of a non-naturally occurring BaNT enzymatic domainvariant of SEQ ID NO: 23, such as, e.g., a conservative BaNT enzymaticdomain variant, a non-conservative BaNT enzymatic domain variant, anactive BaNT enzymatic domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BaNT enzymatic domaincomprises amino acids 1/2-420 of a non-naturally occurring BaNTenzymatic domain variant of SEQ ID NO: 23, such as, e.g., a conservativeBaNT enzymatic domain variant, a non-conservative BaNT enzymatic domainvariant, an active BaNT enzymatic domain fragment, or any combinationthereof.

In other aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 23; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the enzymaticdomain of SEQ ID NO: 23. In yet other aspects of this embodiment, a BaNTenzymatic domain comprises a polypeptide having an amino acid identityof, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% to amino acids 1/2-420 of SEQ ID NO: 23; orat most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or atmost 95% to amino acids 1/2-420 of SEQ ID NO: 23.

In other aspects of this embodiment, a BaNT enzymatic domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the enzymatic domain of SEQ ID NO: 23; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the enzymatic domain of SEQ ID NO: 23. In yet other aspectsof this embodiment, a BaNT enzymatic domain comprises a polypeptidehaving, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-420 of SEQ ID NO: 23; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-420 of SEQ ID NO: 23. In still other aspects of this embodiment, aBaNT enzymatic domain comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the enzymaticdomain of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the enzymatic domain of SEQ ID NO: 23. Infurther other aspects of this embodiment, a BaNT enzymatic domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-420 of SEQID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1/2-420 of SEQ ID NO: 23.

In another embodiment, a Clostridial toxin enzymatic domain comprises aBuNT enzymatic domain. In an aspect of this embodiment, a BuNT enzymaticdomain comprises the enzymatic domains of SEQ ID NO: 24 or SEQ ID NO:25. In other aspects of this embodiment, a BuNT enzymatic domaincomprises amino acids 1/2-411 of SEQ ID NO: 24. In another aspect ofthis embodiment, a BuNT enzymatic domain comprises a naturally occurringBuNT enzymatic domain variant, such as, e.g., an enzymatic domain from aBuNT isoform or an enzymatic domain from a BuNT subtype. In anotheraspect of this embodiment, a BuNT enzymatic domain comprises a naturallyoccurring BuNT enzymatic domain variant of SEQ ID NO: 24 or SEQ ID NO:25, such as, e.g., a BuNT isoform enzymatic domain or a BuNT subtypeenzymatic domain. In another aspect of this embodiment, a BuNT enzymaticdomain comprises amino acids 1/2-411 of a naturally occurring BuNTenzymatic domain variant of SEQ ID NO: 24, such as, e.g., a BuNT isoformenzymatic domain or a BuNT subtype enzymatic domain. In still anotheraspect of this embodiment, a BuNT enzymatic domain comprises anon-naturally occurring BuNT enzymatic domain variant, such as, e.g., aconservative BuNT enzymatic domain variant, a non-conservative BuNTenzymatic domain variant, an active BuNT enzymatic domain fragment, orany combination thereof. In still another aspect of this embodiment, aBuNT enzymatic domain comprises the enzymatic domain of a non-naturallyoccurring BuNT enzymatic domain variant of SEQ ID NO: 24 or SEQ ID NO:25, such as, e.g., a conservative BuNT enzymatic domain variant, anon-conservative BuNT enzymatic domain variant, an active BuNT enzymaticdomain fragment, or any combination thereof. In still another aspect ofthis embodiment, a BuNT enzymatic domain comprises amino acids 1/2-411of a non-naturally occurring BuNT enzymatic domain variant of SEQ ID NO:24, such as, e.g., a conservative BuNT enzymatic domain variant, anon-conservative BuNT enzymatic domain variant, an active BuNT enzymaticdomain fragment, or any combination thereof.

In other aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe enzymatic domain of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%,at most 75%, at most 80%, at most 85%, at most 90%, or at most 95% tothe enzymatic domain of SEQ ID NO: 24 or SEQ ID NO: 25. In yet otheraspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 1/2-411 of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%,at most 75%, at most 80%, at most 85%, at most 90%, or at most 95% toamino acids 1/2-411 of SEQ ID NO: 24 or SEQ ID NO: 25.

In other aspects of this embodiment, a BuNT enzymatic domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the enzymatic domain of SEQ ID NO: 24 or SEQID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the enzymatic domain of SEQ ID NO: 240R SEQ ID NO: 25. Inyet other aspects of this embodiment, a BuNT enzymatic domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to amino acids 1/2-411 of SEQ ID NO: 24 orSEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 1/2-411 of SEQ ID NO: 24 or SEQ IDNO: 25. In still other aspects of this embodiment, a BuNT enzymaticdomain comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to the enzymatic domain of SEQID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the enzymatic domain of SEQ ID NO: 24or SEQ ID NO: 25. In further other aspects of this embodiment, a BuNTenzymatic domain comprises a polypeptide having, e.g., at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids1/2-411 of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 1/2-411 of SEQID NO: 24 or SEQ ID NO: 25.

The “translocation domain” comprises a portion of a Clostridialneurotoxin heavy chain having a translocation activity. By“translocation” is meant the ability to facilitate the transport of apolypeptide through a vesicular membrane, thereby exposing some or allof the polypeptide to the cytoplasm. In the various botulinumneurotoxins translocation is thought to involve an allostericconformational change of the heavy chain caused by a decrease in pHwithin the endosome. This conformational change appears to involve andbe mediated by the N terminal half of the heavy chain and to result inthe formation of pores in the vesicular membrane; this change permitsthe movement of the proteolytic light chain from within the endosomalvesicle into the cytoplasm. See e.g., Lacy, et al., Nature Struct. Biol.5:898-902 (October 1998).

The amino acid sequence of the translocation-mediating portion of thebotulinum neurotoxin heavy chain is known to those of skill in the art;additionally, those amino acid residues within this portion that areknown to be essential for conferring the translocation activity are alsoknown. It would therefore be well within the ability of one of ordinaryskill in the art, for example, to employ the naturally occurringN-terminal peptide half of the heavy chain of any of the variousClostridium tetanus or Clostridium botulinum neurotoxin subtypes as atranslocation domain, or to design an analogous translocation domain byaligning the primary sequences of the N-terminal halves of the variousheavy chains and selecting a consensus primary translocation sequencebased on conserved amino acid, polarity, steric and hydrophobicitycharacteristics between the sequences.

Aspects of the present specification provide, in part, a Clostridialtoxin translocation domain. As used herein, the term “Clostridial toxintranslocation domain” refers to any Clostridial toxin polypeptide thatcan execute the translocation step of the intoxication process thatmediates Clostridial toxin light chain translocation. Thus, aClostridial toxin translocation domain facilitates the movement of aClostridial toxin light chain across a membrane and encompasses themovement of a Clostridial toxin light chain through the membrane anintracellular vesicle into the cytoplasm of a cell. Non-limitingexamples of a Clostridial toxin translocation domain include, e.g., aBoNT/A translocation domain, a BoNT/B translocation domain, a BoNT/C1translocation domain, a BoNT/D translocation domain, a BoNT/Etranslocation domain, a BoNT/F translocation domain, a BoNT/Gtranslocation domain, a TeNT translocation domain, a BaNT translocationdomain, and a BuNT translocation domain.

A Clostridial toxin translocation domain includes, without limitation,naturally occurring Clostridial toxin translocation domain variants,such as, e.g., Clostridial toxin translocation domain isoforms andClostridial toxin translocation domain subtypes; non-naturally occurringClostridial toxin translocation domain variants, such as, e.g.,conservative Clostridial toxin translocation domain variants,non-conservative Clostridial toxin translocation domain variants, activeClostridial toxin translocation domain fragments thereof, or anycombination thereof.

As used herein, the term “Clostridial toxin translocation domainvariant,” whether naturally-occurring or non-naturally-occurring, refersto a Clostridial toxin translocation domain that has at least one aminoacid change from the corresponding region of the disclosed referencesequences (Table 1) and can be described in percent identity to thecorresponding region of that reference sequence. Unless expresslyindicated, Clostridial toxin translocation domain variants useful topractice disclosed embodiments are variants that execute thetranslocation step of the intoxication process that mediates Clostridialtoxin light chain translocation. As non-limiting examples, a BoNT/Atranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 455-873 of SEQ ID NO: 1; a BoNT/Btranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 447-860 of SEQ ID NO: 6; a BoNT/C1translocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 454-868 of SEQ ID NO: 11; a BoNT/Dtranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 451-864 of SEQ ID NO: 13; a BoNT/Etranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 427-847 of SEQ ID NO: 15; a BoNT/Ftranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 446-865 of SEQ ID NO: 18; a BoNT/Gtranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 451-865 of SEQ ID NO: 21; a TeNTtranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 468-881 of SEQ ID NO: 22; a BaNTtranslocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 436-857 of SEQ ID NO: 23; and aBuNT translocation domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 427-847 of SEQ ID NO: 24.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxintranslocation domain variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3, BoNT/A4, and BoNT/A5, with specific translocation domainsubtypes showing about 85-87% amino acid identity when compared to theBoNT/A translocation domain subtype of SEQ ID NO: 1. As used herein, theterm “naturally occurring Clostridial toxin translocation domainvariant” refers to any Clostridial toxin translocation domain producedby a naturally-occurring process, including, without limitation,Clostridial toxin translocation domain isoforms produced fromalternatively-spliced transcripts, Clostridial toxin translocationdomain isoforms produced by spontaneous mutation and Clostridial toxintranslocation domain subtypes. A naturally occurring Clostridial toxintranslocation domain variant can function in substantially the samemanner as the reference Clostridial toxin translocation domain on whichthe naturally occurring Clostridial toxin translocation domain variantis based, and can be substituted for the reference Clostridial toxintranslocation domain in any aspect of the present specification.

A non-limiting examples of a naturally occurring Clostridial toxintranslocation domain variant is a Clostridial toxin translocation domainisoform such as, e.g., a BoNT/A translocation domain isoform, a BoNT/Btranslocation domain isoform, a BoNT/C1 translocation domain isoform, aBoNT/D translocation domain isoform, a BoNT/E translocation domainisoform, a BoNT/F translocation domain isoform, a BoNT/G translocationdomain isoform, a TeNT translocation domain isoform, a BaNTtranslocation domain isoform, and a BuNT translocation domain isoform.Another non-limiting examples of a naturally occurring Clostridial toxintranslocation domain variant is a Clostridial toxin translocation domainsubtype such as, e.g., a translocation domain from subtype BoNT/A1,BoNT/A2, BoNT/A3, BoNT/A4, and BoNT/A5; a translocation domain fromsubtype BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/B nonproteolytic; atranslocation domain from subtype BoNT/C1-1 and BoNT/C1-2; atranslocation domain from subtype BoNT/E1, BoNT/E2 and BoNT/E3; atranslocation domain from subtype BoNT/F1, BoNT/F2, BoNT/F3; and atranslocation domain from subtype BuNT-1 and BuNT-2.

As used herein, the term “non-naturally occurring Clostridial toxintranslocation domain variant” refers to any Clostridial toxintranslocation domain produced with the aid of human manipulation,including, without limitation, Clostridial toxin translocation domainsproduced by genetic engineering using random mutagenesis or rationaldesign and Clostridial toxin translocation domains produced by chemicalsynthesis. Non-limiting examples of non-naturally occurring Clostridialtoxin translocation domain variants include, e.g., conservativeClostridial toxin translocation domain variants, non-conservativeClostridial toxin translocation domain variants, and active Clostridialtoxin translocation domain fragments.

As used herein, the term “conservative Clostridial toxin translocationdomain variant” refers to a Clostridial toxin translocation domain thathas at least one amino acid substituted by another amino acid or anamino acid analog that has at least one property similar to that of theoriginal amino acid from the reference Clostridial toxin translocationdomain sequence (Table 1). Examples of properties include, withoutlimitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative Clostridial toxin translocationdomain variant can function in substantially the same manner as thereference Clostridial toxin translocation domain on which theconservative Clostridial toxin translocation domain variant is based,and can be substituted for the reference Clostridial toxin translocationdomain in any aspect of the present specification. Non-limiting examplesof a conservative Clostridial toxin translocation domain variantinclude, e.g., conservative BoNT/A translocation domain variants,conservative BoNT/B translocation domain variants, conservative BoNT/C1translocation domain variants, conservative BoNT/D translocation domainvariants, conservative BoNT/E translocation domain variants,conservative BoNT/F translocation domain variants, conservative BoNT/Gtranslocation domain variants, conservative TeNT translocation domainvariants, conservative BaNT translocation domain variants, andconservative BuNT translocation domain variants.

As used herein, the term “non-conservative Clostridial toxintranslocation domain variant” refers to a Clostridial toxintranslocation domain in which 1) at least one amino acid is deleted fromthe reference Clostridial toxin translocation domain on which thenon-conservative Clostridial toxin translocation domain variant isbased; 2) at least one amino acid added to the reference Clostridialtoxin translocation domain on which the non-conservative Clostridialtoxin translocation domain is based; or 3) at least one amino acid issubstituted by another amino acid or an amino acid analog that does notshare any property similar to that of the original amino acid from thereference Clostridial toxin translocation domain sequence (Table 1). Anon-conservative Clostridial toxin translocation domain variant canfunction in substantially the same manner as the reference Clostridialtoxin translocation domain on which the non-conservative Clostridialtoxin translocation domain variant is based, and can be substituted forthe reference Clostridial toxin translocation domain in any aspect ofthe present specification. Non-limiting examples of a non-conservativeClostridial toxin translocation domain variant include, e.g.,non-conservative BoNT/A translocation domain variants, non-conservativeBoNT/B translocation domain variants, non-conservative BoNT/C1translocation domain variants, non-conservative BoNT/D translocationdomain variants, non-conservative BoNT/E translocation domain variants,non-conservative BoNT/F translocation domain variants, non-conservativeBoNT/G translocation domain variants, and non-conservative TeNTtranslocation domain variants, non-conservative BaNT translocationdomain variants, and non-conservative BuNT translocation domainvariants.

As used herein, the term “active Clostridial toxin translocation domainfragment” refers to any of a variety of Clostridial toxin fragmentscomprising the translocation domain can be useful in aspects of thepresent specification with the proviso that these active fragments canfacilitate the release of the LC from intracellular vesicles into thecytoplasm of the target cell and thus participate in executing theoverall cellular mechanism whereby a Clostridial toxin proteolyticallycleaves a substrate. The translocation domains from the heavy chains ofClostridial toxins are approximately 410-430 amino acids in length andcomprise a translocation domain (Table 1). Research has shown that theentire length of a translocation domain from a Clostridial toxin heavychain is not necessary for the translocating activity of thetranslocation domain. Thus, aspects of this embodiment include aClostridial toxin translocation domain having a length of, e.g., atleast 350, 375, 400, or 425 amino acids. Other aspects of thisembodiment include a Clostridial toxin translocation domain having alength of, e.g., at most 350, 375, 400, or 425 amino acids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin translocationdomain variants and non-naturally-occurring Clostridial toxintranslocation domain variants, including, without limitation, globalmethods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin translocationdomain. In an aspect of this embodiment, a Clostridial toxintranslocation domain comprises a naturally occurring Clostridial toxintranslocation domain variant, such as, e.g., a Clostridial toxintranslocation domain isoform or a Clostridial toxin translocation domainsubtype. In another aspect of this embodiment, a Clostridial toxintranslocation domain comprises a non-naturally occurring Clostridialtoxin translocation domain variant, such as, e.g., a conservativeClostridial toxin translocation domain variant, a non-conservativeClostridial toxin translocation domain variant, an active Clostridialtoxin translocation domain fragment, or any combination thereof.

In another embodiment, a hydrophobic amino acid at one particularposition in the polypeptide chain of the Clostridial toxin translocationdomain can be substituted with another hydrophobic amino acid. Examplesof hydrophobic amino acids include, e.g., C, F, I, L, M, V and W. Inanother aspect of this embodiment, an aliphatic amino acid at oneparticular position in the polypeptide chain of the Clostridial toxintranslocation domain can be substituted with another aliphatic aminoacid. Examples of aliphatic amino acids include, e.g., A, I, L, P, andV. In yet another aspect of this embodiment, an aromatic amino acid atone particular position in the polypeptide chain of the Clostridialtoxin translocation domain can be substituted with another aromaticamino acid. Examples of aromatic amino acids include, e.g., F, H, W andY. In still another aspect of this embodiment, a stacking amino acid atone particular position in the polypeptide chain of the Clostridialtoxin translocation domain can be substituted with another stackingamino acid. Examples of stacking amino acids include, e.g., F, H, W andY. In a further aspect of this embodiment, a polar amino acid at oneparticular position in the polypeptide chain of the Clostridial toxintranslocation domain can be substituted with another polar amino acid.Examples of polar amino acids include, e.g., D, E, K, N, Q, and R. In afurther aspect of this embodiment, a less polar or indifferent aminoacid at one particular position in the polypeptide chain of theClostridial toxin translocation domain can be substituted with anotherless polar or indifferent amino acid. Examples of less polar orindifferent amino acids include, e.g., A, H, G, P, S, T, and Y. In a yetfurther aspect of this embodiment, a positive charged amino acid at oneparticular position in the polypeptide chain of the Clostridial toxintranslocation domain can be substituted with another positive chargedamino acid. Examples of positive charged amino acids include, e.g., K,R, and H. In a still further aspect of this embodiment, a negativecharged amino acid at one particular position in the polypeptide chainof the Clostridial toxin translocation domain can be substituted withanother negative charged amino acid. Examples of negative charged aminoacids include, e.g., D and E. In another aspect of this embodiment, asmall amino acid at one particular position in the polypeptide chain ofthe Clostridial toxin translocation domain can be substituted withanother small amino acid. Examples of small amino acids include, e.g.,A, D, G, N, P, S, and T. In yet another aspect of this embodiment, aC-beta branching amino acid at one particular position in thepolypeptide chain of the Clostridial toxin translocation domain can besubstituted with another C-beta branching amino acid. Examples of C-betabranching amino acids include, e.g., I, T and V.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/A translocation domain. In an aspect of thisembodiment, a BoNT/A translocation domain comprises the translocationdomains of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, orSEQ ID NO: 5. In other aspects of this embodiment, a BoNT/Atranslocation domain comprises amino acids 455-873 of SEQ ID NO: 1. Inanother aspect of this embodiment, a BoNT/A translocation domaincomprises a naturally occurring BoNT/A translocation domain variant,such as, e.g., an translocation domain from a BoNT/A isoform or antranslocation domain from a BoNT/A subtype. In another aspect of thisembodiment, a BoNT/A translocation domain comprises a naturallyoccurring BoNT/A translocation domain variant of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., aBoNT/A isoform translocation domain or a BoNT/A subtype translocationdomain. In another aspect of this embodiment, a BoNT/A translocationdomain comprises amino acids 455-873 of a naturally occurring BoNT/Atranslocation domain variant of SEQ ID NO: 1, such as, e.g., a BoNT/Aisoform translocation domain or a BoNT/A subtype translocation domain.In still another aspect of this embodiment, a BoNT/A translocationdomain comprises a non-naturally occurring BoNT/A translocation domainvariant, such as, e.g., a conservative BoNT/A translocation domainvariant, a non-conservative BoNT/A translocation domain variant, anactive BoNT/A translocation domain fragment, or any combination thereof.In still another aspect of this embodiment, a BoNT/A translocationdomain comprises the translocation domain of a non-naturally occurringBoNT/A translocation domain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as, e.g., a conservativeBoNT/A translocation domain variant, a non-conservative BoNT/Atranslocation domain variant, an active BoNT/A translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/A translocation domain comprises amino acids 455-873of a non-naturally occurring BoNT/A translocation domain variant of SEQID NO: 1, such as, e.g., a conservative BoNT/A translocation domainvariant, a non-conservative BoNT/A translocation domain variant, anactive BoNT/A translocation domain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/A translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the translocationdomain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQID NO: 5. In yet other aspects of this embodiment, a BoNT/Atranslocation domain comprises a polypeptide having an amino acididentity of, e.g., at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% to amino acids 455-873 of SEQ ID NO:1; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,or at most 95% to amino acids 455-873 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5;or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the translocation domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In yet other aspects of thisembodiment, a BoNT/A translocation domain comprises a polypeptidehaving, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 455-873 of SEQ ID NO: 1; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids455-873 of SEQ ID NO: 1. In still other aspects of this embodiment, aBoNT/A translocation domain comprises a polypeptide having, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to thetranslocation domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In further otheraspects of this embodiment, a BoNT/A translocation domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 455-873 of SEQ ID NO: 1; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 455-873 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/B translocation domain. In an aspect of thisembodiment, a BoNT/B translocation domain comprises the translocationdomains of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, orSEQ ID NO: 10. In other aspects of this embodiment, a BoNT/Btranslocation domain comprises amino acids 447-860 of SEQ ID NO: 6. Inanother aspect of this embodiment, a BoNT/B translocation domaincomprises a naturally occurring BoNT/B translocation domain variant,such as, e.g., an translocation domain from a BoNT/B isoform or antranslocation domain from a BoNT/B subtype. In another aspect of thisembodiment, a BoNT/B translocation domain comprises a naturallyoccurring BoNT/B translocation domain variant of SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., aBoNT/B isoform translocation domain or a BoNT/B subtype translocationdomain. In another aspect of this embodiment, a BoNT/B translocationdomain comprises amino acids 447-860 of a naturally occurring BoNT/Btranslocation domain variant of SEQ ID NO: 6, such as, e.g., a BoNT/Bisoform translocation domain or a BoNT/B subtype translocation domain.In still another aspect of this embodiment, a BoNT/B translocationdomain comprises a non-naturally occurring BoNT/B translocation domainvariant, such as, e.g., a conservative BoNT/B translocation domainvariant, a non-conservative BoNT/B translocation domain variant, anactive BoNT/B translocation domain fragment, or any combination thereof.In still another aspect of this embodiment, a BoNT/B translocationdomain comprises the translocation domain of a non-naturally occurringBoNT/B translocation domain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as, e.g., a conservativeBoNT/B translocation domain variant, a non-conservative BoNT/Btranslocation domain variant, an active BoNT/B translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/B translocation domain comprises amino acids 447-860of a non-naturally occurring BoNT/B translocation domain variant of SEQID NO: 6, such as, e.g., a conservative BoNT/B translocation domainvariant, a non-conservative BoNT/B translocation domain variant, anactive BoNT/B translocation domain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the translocationdomain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQID NO: 10. In yet other aspects of this embodiment, a BoNT/Btranslocation domain comprises a polypeptide having an amino acididentity of, e.g., at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% to amino acids 447-860 of SEQ ID NO:6; or at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,or at most 95% to amino acids 447-860 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/B translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the translocation domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In yet other aspects of thisembodiment, a BoNT/B translocation domain comprises a polypeptidehaving, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 447-860 of SEQ ID NO: 6; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids447-860 of SEQ ID NO: 6. In still other aspects of this embodiment, aBoNT/B translocation domain comprises a polypeptide having, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to thetranslocation domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In further otheraspects of this embodiment, a BoNT/B translocation domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 447-860 of SEQ ID NO: 6; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 447-860 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/C1 translocation domain. In an aspect of thisembodiment, a BoNT/C1 translocation domain comprises the translocationdomains of SEQ ID NO: 11 or SEQ ID NO: 12. In other aspects of thisembodiment, a BoNT/C1 translocation domain comprises amino acids 454-868of SEQ ID NO: 11. In another aspect of this embodiment, a BoNT/C1translocation domain comprises a naturally occurring BoNT/C1translocation domain variant, such as, e.g., an translocation domainfrom a BoNT/C1 isoform or an translocation domain from a BoNT/C1subtype. In another aspect of this embodiment, a BoNT/C1 translocationdomain comprises a naturally occurring BoNT/C1 translocation domainvariant of SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., a BoNT/C1isoform translocation domain or a BoNT/C1 subtype translocation domain.In another aspect of this embodiment, a BoNT/C1 translocation domaincomprises amino acids 454-868 of a naturally occurring BoNT/C1translocation domain variant of SEQ ID NO: 11, such as, e.g., a BoNT/C1isoform translocation domain or a BoNT/C1 subtype translocation domain.In still another aspect of this embodiment, a BoNT/C1 translocationdomain comprises a non-naturally occurring BoNT/C1 translocation domainvariant, such as, e.g., a conservative BoNT/C1 translocation domainvariant, a non-conservative BoNT/C1 translocation domain variant, anactive BoNT/C1 translocation domain fragment, or any combinationthereof. In still another aspect of this embodiment, a BoNT/C1translocation domain comprises the translocation domain of anon-naturally occurring BoNT/C1 translocation domain variant of SEQ IDNO: 11 or SEQ ID NO: 12, such as, e.g., a conservative BoNT/C1translocation domain variant, a non-conservative BoNT/C1 translocationdomain variant, an active BoNT/C1 translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/C1 translocation domain comprises amino acids 454-868 of anon-naturally occurring BoNT/C1 translocation domain variant of SEQ IDNO: 11, such as, e.g., a conservative BoNT/C1 translocation domainvariant, a non-conservative BoNT/C1 translocation domain variant, anactive BoNT/C1 translocation domain fragment, or any combinationthereof.

In other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 11 or SEQ ID NO: 12; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to the translocation domain of SEQ ID NO: 11 or SEQ ID NO: 12. Inyet other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to amino acids 454-868 of SEQ ID NO: 11; or at most 70%, at most75%, at most 80%, at most 85%, at most 90%, or at most 95% to aminoacids 454-868 of SEQ ID NO: 11.

In other aspects of this embodiment, a BoNT/C1 translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 11 or SEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 11 or SEQ ID NO: 12. In yet other aspects of this embodiment,a BoNT/C1 translocation domain comprises a polypeptide having, e.g., atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 454-868 of SEQ ID NO: 11; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids454-868 of SEQ ID NO: 11. In still other aspects of this embodiment, aBoNT/C1 translocation domain comprises a polypeptide having, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to thetranslocation domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the translocationdomain of SEQ ID NO: 11 or SEQ ID NO: 12. In further other aspects ofthis embodiment, a BoNT/C1 translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 454-868 of SEQ ID NO: 11; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids454-868 of SEQ ID NO: 11.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/D translocation domain. In an aspect of thisembodiment, a BoNT/D translocation domain comprises the translocationdomains of SEQ ID NO: 13 or SEQ ID NO: 14. In other aspects of thisembodiment, a BoNT/D translocation domain comprises amino acids 451-864of SEQ ID NO: 13. In another aspect of this embodiment, a BoNT/Dtranslocation domain comprises a naturally occurring BoNT/Dtranslocation domain variant, such as, e.g., an translocation domainfrom a BoNT/D isoform or an translocation domain from a BoNT/D subtype.In another aspect of this embodiment, a BoNT/D translocation domaincomprises a naturally occurring BoNT/D translocation domain variant ofSEQ ID NO: 13 or SEQ ID NO: 14, such as, e.g., a BoNT/D isoformtranslocation domain or a BoNT/D subtype translocation domain. Inanother aspect of this embodiment, a BoNT/D translocation domaincomprises amino acids 451-864 of a naturally occurring BoNT/Dtranslocation domain variant of SEQ ID NO: 13, such as, e.g., a BoNT/Disoform translocation domain or a BoNT/D subtype translocation domain.In still another aspect of this embodiment, a BoNT/D translocationdomain comprises a non-naturally occurring BoNT/D translocation domainvariant, such as, e.g., a conservative BoNT/D translocation domainvariant, a non-conservative BoNT/D translocation domain variant, anactive BoNT/D translocation domain fragment, or any combination thereof.In still another aspect of this embodiment, a BoNT/D translocationdomain comprises the translocation domain of a non-naturally occurringBoNT/D translocation domain variant of SEQ ID NO: 13 or SEQ ID NO: 14,such as, e.g., a conservative BoNT/D translocation domain variant, anon-conservative BoNT/D translocation domain variant, an active BoNT/Dtranslocation domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/D translocation domaincomprises amino acids 451-864 of a non-naturally occurring BoNT/Dtranslocation domain variant of SEQ ID NO: 13, such as, e.g., aconservative BoNT/D translocation domain variant, a non-conservativeBoNT/D translocation domain variant, an active BoNT/D translocationdomain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 13 or SEQ ID NO: 14; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to the translocation domain of SEQ ID NO: 13 or SEQ ID NO: 14. Inyet other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to amino acids 451-864 of SEQ ID NO: 13; or at most 70%, at most75%, at most 80%, at most 85%, at most 90%, or at most 95% to aminoacids 451-864 of SEQ ID NO: 13.

In other aspects of this embodiment, a BoNT/D translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 13 or SEQ ID NO: 14; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 13 or SEQ ID NO: 14. In yet other aspects of this embodiment,a BoNT/D translocation domain comprises a polypeptide having, e.g., atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 451-864 of SEQ ID NO: 13; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids451-864 of SEQ ID NO: 13. In still other aspects of this embodiment, aBoNT/D translocation domain comprises a polypeptide having, e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to thetranslocation domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the translocationdomain of SEQ ID NO: 13 or SEQ ID NO: 14. In further other aspects ofthis embodiment, a BoNT/D translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 451-864 of SEQ ID NO: 13; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids451-864 of SEQ ID NO: 13.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/E translocation domain. In an aspect of thisembodiment, a BoNT/E translocation domain comprises the translocationdomains of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In otheraspects of this embodiment, a BoNT/E translocation domain comprisesamino acids 427-847 of SEQ ID NO: 15. In another aspect of thisembodiment, a BoNT/E translocation domain comprises a naturallyoccurring BoNT/E translocation domain variant, such as, e.g., antranslocation domain from a BoNT/E isoform or an translocation domainfrom a BoNT/E subtype. In another aspect of this embodiment, a BoNT/Etranslocation domain comprises a naturally occurring BoNT/Etranslocation domain variant of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ IDNO: 17, such as, e.g., a BoNT/E isoform translocation domain or a BoNT/Esubtype translocation domain. In another aspect of this embodiment, aBoNT/E translocation domain comprises amino acids 427-847 of a naturallyoccurring BoNT/E translocation domain variant of SEQ ID NO: 15, such as,e.g., a BoNT/E isoform translocation domain or a BoNT/E subtypetranslocation domain. In still another aspect of this embodiment, aBoNT/E translocation domain comprises a non-naturally occurring BoNT/Etranslocation domain variant, such as, e.g., a conservative BoNT/Etranslocation domain variant, a non-conservative BoNT/E translocationdomain variant, an active BoNT/E translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/E translocation domain comprises the translocation domain of anon-naturally occurring BoNT/E translocation domain variant of SEQ IDNO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a conservativeBoNT/E translocation domain variant, a non-conservative BoNT/Etranslocation domain variant, an active BoNT/E translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/E translocation domain comprises amino acids 427-847of a non-naturally occurring BoNT/E translocation domain variant of SEQID NO: 15, such as, e.g., a conservative BoNT/E translocation domainvariant, a non-conservative BoNT/E translocation domain variant, anactive BoNT/E translocation domain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQID NO: 17; or at most 70%, at most 75%, at most 80%, at most 85%, atmost 90%, or at most 95% to the translocation domain of SEQ ID NO: 15,SEQ ID NO: 16, or SEQ ID NO: 17. In yet other aspects of thisembodiment, a BoNT/E translocation domain comprises a polypeptide havingan amino acid identity of, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% to amino acids 427-847of SEQ ID NO: 15; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to amino acids 427-847 of SEQ ID NO: 15.

In other aspects of this embodiment, a BoNT/E translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to the translocationdomain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In yet otheraspects of this embodiment, a BoNT/E translocation domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 427-847 of SEQ ID NO: 15; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 427-847 of SEQ ID NO: 15. In still other aspectsof this embodiment, a BoNT/E translocation domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 15, SEQID NO: 16, or SEQ ID NO: 17; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the translocation domain of SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO: 17. In further other aspects of thisembodiment, a BoNT/E translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 427-847 of SEQ ID NO: 15; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids427-847 of SEQ ID NO: 15.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/F translocation domain. In an aspect of thisembodiment, a BoNT/F translocation domain comprises the translocationdomains of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In otheraspects of this embodiment, a BoNT/F translocation domain comprisesamino acids 446-865 of SEQ ID NO: 18. In another aspect of thisembodiment, a BoNT/F translocation domain comprises a naturallyoccurring BoNT/F translocation domain variant, such as, e.g., antranslocation domain from a BoNT/F isoform or an translocation domainfrom a BoNT/F subtype. In another aspect of this embodiment, a BoNT/Ftranslocation domain comprises a naturally occurring BoNT/Ftranslocation domain variant of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ IDNO: 20, such as, e.g., a BoNT/F isoform translocation domain or a BoNT/Fsubtype translocation domain. In another aspect of this embodiment, aBoNT/F translocation domain comprises amino acids 446-865 of a naturallyoccurring BoNT/F translocation domain variant of SEQ ID NO: 18, such as,e.g., a BoNT/F isoform translocation domain or a BoNT/F subtypetranslocation domain. In still another aspect of this embodiment, aBoNT/F translocation domain comprises a non-naturally occurring BoNT/Ftranslocation domain variant, such as, e.g., a conservative BoNT/Ftranslocation domain variant, a non-conservative BoNT/F translocationdomain variant, an active BoNT/F translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/F translocation domain comprises the translocation domain of anon-naturally occurring BoNT/F translocation domain variant of SEQ IDNO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a conservativeBoNT/F translocation domain variant, a non-conservative BoNT/Ftranslocation domain variant, an active BoNT/F translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/F translocation domain comprises amino acids 446-865of a non-naturally occurring BoNT/F translocation domain variant of SEQID NO: 18, such as, e.g., a conservative BoNT/F translocation domainvariant, a non-conservative BoNT/F translocation domain variant, anactive BoNT/F translocation domain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQID NO: 20; or at most 70%, at most 75%, at most 80%, at most 85%, atmost 90%, or at most 95% to the translocation domain of SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20. In yet other aspects of thisembodiment, a BoNT/F translocation domain comprises a polypeptide havingan amino acid identity of, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% to amino acids 446-865of SEQ ID NO: 18; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to amino acids 446-865 of SEQ ID NO: 18.

In other aspects of this embodiment, a BoNT/F translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to the translocationdomain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In yet otheraspects of this embodiment, a BoNT/F translocation domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 446-865 of SEQ ID NO: 18; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 446-865 of SEQ ID NO: 18. In still other aspectsof this embodiment, a BoNT/F translocation domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 18, SEQID NO: 19, or SEQ ID NO: 20; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the translocation domain of SEQ ID NO:18, SEQ ID NO: 19, or SEQ ID NO: 20. In further other aspects of thisembodiment, a BoNT/F translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 446-865 of SEQ ID NO: 18; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids446-865 of SEQ ID NO: 18.

In another embodiment, a Clostridial toxin translocation domaincomprises a BoNT/G translocation domain. In an aspect of thisembodiment, a BoNT/G translocation domain comprises the translocationdomains of SEQ ID NO: 21. In other aspects of this embodiment, a BoNT/Gtranslocation domain comprises amino acids 451-865 of SEQ ID NO: 21. Inanother aspect of this embodiment, a BoNT/G translocation domaincomprises a naturally occurring BoNT/G translocation domain variant,such as, e.g., an translocation domain from a BoNT/G isoform or antranslocation domain from a BoNT/G subtype. In another aspect of thisembodiment, a BoNT/G translocation domain comprises a naturallyoccurring BoNT/G translocation domain variant of SEQ ID NO: 21, such as,e.g., a BoNT/G isoform translocation domain or a BoNT/G subtypetranslocation domain. In another aspect of this embodiment, a BoNT/Gtranslocation domain comprises amino acids 451-865 of a naturallyoccurring BoNT/G translocation domain variant of SEQ ID NO: 21, such as,e.g., a BoNT/G isoform translocation domain or a BoNT/G subtypetranslocation domain. In still another aspect of this embodiment, aBoNT/G translocation domain comprises a non-naturally occurring BoNT/Gtranslocation domain variant, such as, e.g., a conservative BoNT/Gtranslocation domain variant, a non-conservative BoNT/G translocationdomain variant, an active BoNT/G translocation domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/G translocation domain comprises the translocation domain of anon-naturally occurring BoNT/G translocation domain variant of SEQ IDNO: 21, such as, e.g., a conservative BoNT/G translocation domainvariant, a non-conservative BoNT/G translocation domain variant, anactive BoNT/G translocation domain fragment, or any combination thereof.In still another aspect of this embodiment, a BoNT/G translocationdomain comprises amino acids 451-865 of a non-naturally occurring BoNT/Gtranslocation domain variant of SEQ ID NO: 21, such as, e.g., aconservative BoNT/G translocation domain variant, a non-conservativeBoNT/G translocation domain variant, an active BoNT/G translocationdomain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 21; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to thetranslocation domain of SEQ ID NO: 21. In yet other aspects of thisembodiment, a BoNT/G translocation domain comprises a polypeptide havingan amino acid identity of, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% to amino acids 451-865of SEQ ID NO: 21; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to amino acids 451-865 of SEQ ID NO: 21.

In other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 21. Inyet other aspects of this embodiment, a BoNT/G translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 451-865 of SEQID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 451-865 of SEQ ID NO: 21. In still other aspectsof this embodiment, a BoNT/G translocation domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 21; orat most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to thetranslocation domain of SEQ ID NO: 21. In further other aspects of thisembodiment, a BoNT/G translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 451-865 of SEQ ID NO: 21; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids451-865 of SEQ ID NO: 21.

In another embodiment, a Clostridial toxin translocation domaincomprises a TeNT translocation domain. In an aspect of this embodiment,a TeNT translocation domain comprises the translocation domains of SEQID NO: 22. In other aspects of this embodiment, a TeNT translocationdomain comprises amino acids 468-881 of SEQ ID NO: 22. In another aspectof this embodiment, a TeNT translocation domain comprises a naturallyoccurring TeNT translocation domain variant, such as, e.g., antranslocation domain from a TeNT isoform or an translocation domain froma TeNT subtype. In another aspect of this embodiment, a TeNTtranslocation domain comprises a naturally occurring TeNT translocationdomain variant of SEQ ID NO: 22, such as, e.g., a TeNT isoformtranslocation domain or a TeNT subtype translocation domain. In anotheraspect of this embodiment, a TeNT translocation domain comprises aminoacids 468-881 of a naturally occurring TeNT translocation domain variantof SEQ ID NO: 22, such as, e.g., a TeNT isoform translocation domain ora TeNT subtype translocation domain. In still another aspect of thisembodiment, a TeNT translocation domain comprises a non-naturallyoccurring TeNT translocation domain variant, such as, e.g., aconservative TeNT translocation domain variant, a non-conservative TeNTtranslocation domain variant, an active TeNT translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a TeNT translocation domain comprises the translocationdomain of a non-naturally occurring TeNT translocation domain variant ofSEQ ID NO: 22, such as, e.g., a conservative TeNT translocation domainvariant, a non-conservative TeNT translocation domain variant, an activeTeNT translocation domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a TeNT translocation domain comprisesamino acids 468-881 of a non-naturally occurring TeNT translocationdomain variant of SEQ ID NO: 22, such as, e.g., a conservative TeNTtranslocation domain variant, a non-conservative TeNT translocationdomain variant, an active TeNT translocation domain fragment, or anycombination thereof.

In other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 22; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to thetranslocation domain of SEQ ID NO: 22. In yet other aspects of thisembodiment, a TeNT translocation domain comprises a polypeptide havingan amino acid identity of, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% to amino acids 468-881of SEQ ID NO: 22; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to amino acids 468-881 of SEQ ID NO: 22.

In other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 22. Inyet other aspects of this embodiment, a TeNT translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 468-881 of SEQID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 468-881 of SEQ ID NO: 22. In still other aspectsof this embodiment, a TeNT translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to the translocation domain of SEQ ID NO: 22; or at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the translocationdomain of SEQ ID NO: 22. In further other aspects of this embodiment, aTeNT translocation domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to amino acids468-881 of SEQ ID NO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 468-881 of SEQ ID NO: 22.

In another embodiment, a Clostridial toxin translocation domaincomprises a BaNT translocation domain. In an aspect of this embodiment,a BaNT translocation domain comprises the translocation domains of SEQID NO: 23. In other aspects of this embodiment, a BaNT translocationdomain comprises amino acids 436-857 of SEQ ID NO: 23. In another aspectof this embodiment, a BaNT translocation domain comprises a naturallyoccurring BaNT translocation domain variant, such as, e.g., antranslocation domain from a BaNT isoform or an translocation domain froma BaNT subtype. In another aspect of this embodiment, a BaNTtranslocation domain comprises a naturally occurring BaNT translocationdomain variant of SEQ ID NO: 23, such as, e.g., a BaNT isoformtranslocation domain or a BaNT subtype translocation domain. In anotheraspect of this embodiment, a BaNT translocation domain comprises aminoacids 436-857 of a naturally occurring BaNT translocation domain variantof SEQ ID NO: 23, such as, e.g., a BaNT isoform translocation domain ora BaNT subtype translocation domain. In still another aspect of thisembodiment, a BaNT translocation domain comprises a non-naturallyoccurring BaNT translocation domain variant, such as, e.g., aconservative BaNT translocation domain variant, a non-conservative BaNTtranslocation domain variant, an active BaNT translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BaNT translocation domain comprises the translocationdomain of a non-naturally occurring BaNT translocation domain variant ofSEQ ID NO: 23, such as, e.g., a conservative BaNT translocation domainvariant, a non-conservative BaNT translocation domain variant, an activeBaNT translocation domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BaNT translocation domain comprisesamino acids 436-857 of a non-naturally occurring BaNT translocationdomain variant of SEQ ID NO: 23, such as, e.g., a conservative BaNTtranslocation domain variant, a non-conservative BaNT translocationdomain variant, an active BaNT translocation domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 23; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to thetranslocation domain of SEQ ID NO: 23. In yet other aspects of thisembodiment, a BaNT translocation domain comprises a polypeptide havingan amino acid identity of, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% to amino acids 436-857of SEQ ID NO: 23; or at most 70%, at most 75%, at most 80%, at most 85%,at most 90%, or at most 95% to amino acids 436-857 of SEQ ID NO: 23.

In other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the translocation domain of SEQ ID NO: 23. Inyet other aspects of this embodiment, a BaNT translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 436-857 of SEQID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 436-857 of SEQ ID NO: 23. In still other aspectsof this embodiment, a BaNT translocation domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to the translocation domain of SEQ ID NO: 23; or at most 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the translocationdomain of SEQ ID NO: 23. In further other aspects of this embodiment, aBaNT translocation domain comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous aminoacid deletions, additions, and/or substitutions relative to amino acids436-857 of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 436-857 of SEQ ID NO: 23.

In another embodiment, a Clostridial toxin translocation domaincomprises a BuNT translocation domain. In an aspect of this embodiment,a BuNT translocation domain comprises the translocation domains of SEQID NO: 24 or SEQ ID NO: 25. In other aspects of this embodiment, a BuNTtranslocation domain comprises amino acids 427-847 of SEQ ID NO: 24. Inanother aspect of this embodiment, a BuNT translocation domain comprisesa naturally occurring BuNT translocation domain variant, such as, e.g.,an translocation domain from a BuNT isoform or an translocation domainfrom a BuNT subtype. In another aspect of this embodiment, a BuNTtranslocation domain comprises a naturally occurring BuNT translocationdomain variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g., a BuNTisoform translocation domain or a BuNT subtype translocation domain. Inanother aspect of this embodiment, a BuNT translocation domain comprisesamino acids 427-847 of a naturally occurring BuNT translocation domainvariant of SEQ ID NO: 24, such as, e.g., a BuNT isoform translocationdomain or a BuNT subtype translocation domain. In still another aspectof this embodiment, a BuNT translocation domain comprises anon-naturally occurring BuNT translocation domain variant, such as,e.g., a conservative BuNT translocation domain variant, anon-conservative BuNT translocation domain variant, an active BuNTtranslocation domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BuNT translocation domain comprisesthe translocation domain of a non-naturally occurring BuNT translocationdomain variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as, e.g., aconservative BuNT translocation domain variant, a non-conservative BuNTtranslocation domain variant, an active BuNT translocation domainfragment, or any combination thereof. In still another aspect of thisembodiment, a BuNT translocation domain comprises amino acids 427-847 ofa non-naturally occurring BuNT translocation domain variant of SEQ IDNO: 24, such as, e.g., a conservative BuNT translocation domain variant,a non-conservative BuNT translocation domain variant, an active BuNTtranslocation domain fragment, or any combination thereof.

In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to the translocation domain of SEQ ID NO: 24 or SEQ ID NO: 25; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to the translocation domain of SEQ ID NO: 24 or SEQ ID NO: 25. Inyet other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO: 25; or at most70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most 95%to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO: 25.

In other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 240R SEQ ID NO: 25. In yet other aspects of this embodiment,a BuNT translocation domain comprises a polypeptide having, e.g., atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO: 25; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 427-847 of SEQ ID NO: 24 or SEQ ID NO: 25. Instill other aspects of this embodiment, a BuNT translocation domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to the translocation domain ofSEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the translocation domain of SEQ ID NO:24 or SEQ ID NO: 25. In further other aspects of this embodiment, a BuNTtranslocation domain comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids427-847 of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 427-847 of SEQID NO: 24 or SEQ ID NO: 25.

Aspects of the present specification provide, in part, a binding domain.As used herein, the term “binding domain” is synonymous with “ligand” or“targeting moiety” and refers to any molecule that can preferentiallyinteract with another molecule present on the surface of a cell underphysiological conditions. The cell surface molecule may comprise apolypeptide, a polysaccharide, a lipid, or may have structuralcharacteristics of more than one of these. As used herein, the term“preferentially interacts” refers to molecule is able to bind its targetreceptor under physiological conditions, or in vitro conditionssubstantially approximating physiological conditions, to a statisticallysignificantly greater degree relative to other, non-target receptor.With reference to a Clostridial toxin binding domain disclosed in thepresent specification, there is a discriminatory binding of theClostridial toxin binding domain to its cognate receptor relative toother receptors. With reference to a non-Clostridial toxin bindingdomain disclosed in the present specification, there is a discriminatorybinding of the non-Clostridial toxin binding domain to it cognatereceptor relative to other receptors.

Thus, in an embodiment, a binding domain that selectively binds a targetreceptor has a dissociation equilibrium constant (K_(D)) that is greaterfor the target receptor relative to a non-target receptor by, e.g., atleast one-fold, at least two-fold, at least three-fold, at least fourfold, at least five-fold, at least 10 fold, at least 50 fold, at least100 fold, at least 1000, at least 10,000, or at least 100,000 fold.

Aspects of the present specification provide, in part, a Clostridialtoxin binding domain. As used herein, the term “Clostridial toxinbinding domain” refers to any Clostridial toxin polypeptide that canexecute the binding step of the intoxication process that initiates theoverall internalization mechanism whereby the modified Clostridial toxindisclosed in the present specification intoxicates a target cell.Non-limiting examples of a Clostridial toxin binding domain include,e.g., a BoNT/A binding domain, a BoNT/B binding domain, a BoNT/C1binding domain, a BoNT/D binding domain, a BoNT/E binding domain, aBoNT/F binding domain, a BoNT/G binding domain, a TeNT binding domain, aBaNT binding domain, and a BuNT binding domain. Other non-limitingexamples of a Clostridial toxin binding domain include, e.g., aminoacids 874-1296 of SEQ ID NO: 1, amino acids 861-1291 of SEQ ID NO: 2,amino acids 869-1291 of SEQ ID NO: 3, amino acids 865-1291 of SEQ ID NO:4, amino acids 848-1252 of SEQ ID NO: 5, amino acids 866-1274 of SEQ IDNO: 6, amino acids 866-1297 of SEQ ID NO: 7, amino acids 882-1315 of SEQID NO: 8, amino acids 858-1268 of SEQ ID NO: 9, and amino acids 848-1251of SEQ ID NO: 10.

A Clostridial toxin binding domain includes, without limitation,naturally occurring Clostridial toxin binding domain variants, such as,e.g., Clostridial toxin binding domain isoforms and Clostridial toxinbinding domain subtypes; non-naturally occurring Clostridial toxinbinding domain variants, such as, e.g., conservative Clostridial toxinbinding domain variants, non-conservative Clostridial toxin bindingdomain variants, active Clostridial toxin binding domain fragmentsthereof, or any combination thereof.

As used herein, the term “Clostridial toxin binding domain variant,”whether naturally-occurring or non-naturally-occurring, refers to aClostridial toxin binding domain that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences(Table 1) and can be described in percent identity to the correspondingregion of that reference sequence. Unless expressly indicated,Clostridial toxin binding domain variants useful to practice disclosedembodiments are variants that execute the translocation step of theintoxication process that mediates Clostridial toxin light chaintranslocation. As non-limiting examples, a BoNT/A binding domain variantwill have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to amino acids874-1296 of SEQ ID NO: 1; a BoNT/B binding domain variant will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to amino acids 861-1291of SEQ ID NO: 6; a BoNT/C1 binding domain variant will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to amino acids 869-1291 of SEQ ID NO:11; a BoNT/D binding domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 865-1291 of SEQ ID NO: 13; a BoNT/Ebinding domain variant will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to amino acids 848-1252 of SEQ ID NO: 15; a BoNT/F bindingdomain variant will have at least one amino acid difference, such as,e.g., an amino acid substitution, deletion or addition, as compared toamino acids 866-1274 of SEQ ID NO: 18; a BoNT/G binding domain variantwill have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to amino acids866-1297 of SEQ ID NO: 21; a TeNT binding domain variant will have atleast one amino acid difference, such as, e.g., an amino acidsubstitution, deletion or addition, as compared to amino acids 882-1315of SEQ ID NO: 22; a BaNT binding domain variant will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to amino acids 858-1268 of SEQ ID NO:23; and a BuNT binding domain variant will have at least one amino aciddifference, such as, e.g., an amino acid substitution, deletion oraddition, as compared to amino acids 848-1251 of SEQ ID NO: 24.

It is recognized by those of skill in the art that within each serotypeof Clostridial toxin there can be naturally occurring Clostridial toxinbinding domain variants that differ somewhat in their amino acidsequence, and also in the nucleic acids encoding these proteins. Forexample, there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2,BoNT/A3, BoNT/A4, and BoNT/A5, with specific binding domain subtypesshowing about 83-97% amino acid identity when compared to the BoNT/Abinding domain subtype of SEQ ID NO: 1. As another example, there arepresently five BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3, BoNT/A4, andBoNT/A5, with specific binding domain subtypes showing about 83-97%amino acid identity when compared to the BoNT/A binding domain subtypeof SEQ ID NO: 1. As used herein, the term “naturally occurringClostridial toxin binding domain variant” refers to any Clostridialtoxin binding domain produced by a naturally-occurring process,including, without limitation, Clostridial toxin binding domain isoformsproduced from alternatively-spliced transcripts, Clostridial toxinbinding domain isoforms produced by spontaneous mutation and Clostridialtoxin binding domain subtypes. A naturally occurring Clostridial toxinbinding domain variant can function in substantially the same manner asthe reference Clostridial toxin binding domain on which the naturallyoccurring Clostridial toxin binding domain variant is based, and can besubstituted for the reference Clostridial toxin binding domain in anyaspect of the present specification.

A non-limiting examples of a naturally occurring Clostridial toxinbinding domain variant is a Clostridial toxin binding domain isoformsuch as, e.g., a BoNT/A binding domain isoform, a BoNT/B binding domainisoform, a BoNT/C1 binding domain isoform, a BoNT/D binding domainisoform, a BoNT/E binding domain isoform, a BoNT/F binding domainisoform, a BoNT/G binding domain isoform, a TeNT binding domain isoform,a BaNT binding domain isoform, and a BuNT binding domain isoform.Another non-limiting examples of a naturally occurring Clostridial toxinbinding domain variant is a Clostridial toxin binding domain subtypesuch as, e.g., a binding domain from subtype BoNT/A1, BoNT/A2, BoNT/A3,BoNT/A4, and BoNT/A5; a binding domain from subtype BoNT/B1, BoNT/B2,BoNT/B bivalent and BoNT/B nonproteolytic; a binding domain from subtypeBoNT/C1-1 and BoNT/C1-2; a binding domain from subtype BoNT/E1, BoNT/E2and BoNT/E3; and a binding domain from subtype BoNT/F1, BoNT/F2, andBoNT/F3; and a binding domain from subtype BuNT-1 and BuNT-2.

As used herein, the term “non-naturally occurring Clostridial toxinbinding domain variant” refers to any Clostridial toxin binding domainproduced with the aid of human manipulation, including, withoutlimitation, Clostridial toxin binding domains produced by geneticengineering using random mutagenesis or rational design and Clostridialtoxin binding domains produced by chemical synthesis. Non-limitingexamples of non-naturally occurring Clostridial toxin binding domainvariants include, e.g., conservative Clostridial toxin binding domainvariants, non-conservative Clostridial toxin binding domain variants,Clostridial toxin binding domain chimeric variants and activeClostridial toxin binding domain fragments.

As used herein, the term “conservative Clostridial toxin binding domainvariant” refers to a Clostridial toxin binding domain that has at leastone amino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference Clostridial toxin binding domain sequence (Table1). Examples of properties include, without limitation, similar size,topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservativeClostridial toxin binding domain variant can function in substantiallythe same manner as the reference Clostridial toxin binding domain onwhich the conservative Clostridial toxin binding domain variant isbased, and can be substituted for the reference Clostridial toxinbinding domain in any aspect of the present specification. Non-limitingexamples of a conservative Clostridial toxin binding domain variantinclude, e.g., conservative BoNT/A binding domain variants, conservativeBoNT/B binding domain variants, conservative BoNT/C1 binding domainvariants, conservative BoNT/D binding domain variants, conservativeBoNT/E binding domain variants, conservative BoNT/F binding domainvariants, conservative BoNT/G binding domain variants, conservative TeNTbinding domain variants, conservative BaNT binding domain variants, andconservative BuNT binding domain variants.

As used herein, the term “non-conservative Clostridial toxin bindingdomain variant” refers to a Clostridial toxin binding domain in which 1)at least one amino acid is deleted from the reference Clostridial toxinbinding domain on which the non-conservative Clostridial toxin bindingdomain variant is based; 2) at least one amino acid added to thereference Clostridial toxin binding domain on which the non-conservativeClostridial toxin binding domain is based; or 3) at least one amino acidis substituted by another amino acid or an amino acid analog that doesnot share any property similar to that of the original amino acid fromthe reference Clostridial toxin binding domain sequence (Table 1). Anon-conservative Clostridial toxin binding domain variant can functionin substantially the same manner as the reference Clostridial toxinbinding domain on which the non-conservative Clostridial toxin bindingdomain variant is based, and can be substituted for the referenceClostridial toxin binding domain in any aspect of the presentspecification. Non-limiting examples of a non-conservative Clostridialtoxin binding domain variant include, e.g., non-conservative BoNT/Abinding domain variants, non-conservative BoNT/B binding domainvariants, non-conservative BoNT/C1 binding domain variants,non-conservative BoNT/D binding domain variants, non-conservative BoNT/Ebinding domain variants, non-conservative BoNT/F binding domainvariants, non-conservative BoNT/G binding domain variants, andnon-conservative TeNT binding domain variants, non-conservative BaNTbinding domain variants, and non-conservative BuNT binding domainvariants.

As used herein, the term “active Clostridial toxin binding domainfragment” refers to any of a variety of Clostridial toxin fragmentscomprising the binding domain can be useful in aspects of the presentspecification with the proviso that these active fragments canfacilitate the release of the LC from intracellular vesicles into thecytoplasm of the target cell and thus participate in executing theoverall cellular mechanism whereby a Clostridial toxin proteolyticallycleaves a substrate. The binding domains from the heavy chains ofClostridial toxins are approximately 400-440 amino acids in length andcomprise a binding domain (Table 1). Research has shown that the entirelength of a binding domain from a Clostridial toxin heavy chain is notnecessary for the translocating activity of the binding domain. Thus,aspects of this embodiment include a Clostridial toxin binding domainhaving a length of, e.g., at least 350, 375, 400, or 425 amino acids.Other aspects of this embodiment include a Clostridial toxin bindingdomain having a length of, e.g., at most 350, 375, 400, or 425 aminoacids.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin binding domainvariants and non-naturally-occurring Clostridial toxin binding domainvariants, including, without limitation, global methods, local methods,and hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a Clostridial toxin binding domain. Inan aspect of this embodiment, a Clostridial toxin binding domaincomprises a naturally occurring Clostridial toxin binding domainvariant, such as, e.g., a Clostridial toxin binding domain isoform or aClostridial toxin binding domain subtype. In another aspect of thisembodiment, a Clostridial toxin binding domain comprises a non-naturallyoccurring Clostridial toxin binding domain variant, such as, e.g., aconservative Clostridial toxin binding domain variant, anon-conservative Clostridial toxin binding domain variant, an activeClostridial toxin binding domain fragment, or any combination thereof.

In another embodiment, a hydrophobic amino acid at one particularposition in the polypeptide chain of the Clostridial toxin bindingdomain can be substituted with another hydrophobic amino acid. Examplesof hydrophobic amino acids include, e.g., C, F, I, L, M, V and W. Inanother aspect of this embodiment, an aliphatic amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinbinding domain can be substituted with another aliphatic amino acid.Examples of aliphatic amino acids include, e.g., A, I, L, P, and V. Inyet another aspect of this embodiment, an aromatic amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinbinding domain can be substituted with another aromatic amino acid.Examples of aromatic amino acids include, e.g., F, H, W and Y. In stillanother aspect of this embodiment, a stacking amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinbinding domain can be substituted with another stacking amino acid.Examples of stacking amino acids include, e.g., F, H, W and Y. In afurther aspect of this embodiment, a polar amino acid at one particularposition in the polypeptide chain of the Clostridial toxin bindingdomain can be substituted with another polar amino acid. Examples ofpolar amino acids include, e.g., D, E, K, N, Q, and R. In a furtheraspect of this embodiment, a less polar or indifferent amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinbinding domain can be substituted with another less polar or indifferentamino acid. Examples of less polar or indifferent amino acids include,e.g., A, H, G, P, S, T, and Y. In a yet further aspect of thisembodiment, a positive charged amino acid at one particular position inthe polypeptide chain of the Clostridial toxin binding domain can besubstituted with another positive charged amino acid. Examples ofpositive charged amino acids include, e.g., K, R, and H. In a stillfurther aspect of this embodiment, a negative charged amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinbinding domain can be substituted with another negative charged aminoacid. Examples of negative charged amino acids include, e.g., D and E.In another aspect of this embodiment, a small amino acid at oneparticular position in the polypeptide chain of the Clostridial toxinbinding domain can be substituted with another small amino acid.Examples of small amino acids include, e.g., A, D, G, N, P, S, and T. Inyet another aspect of this embodiment, a C-beta branching amino acid atone particular position in the polypeptide chain of the Clostridialtoxin binding domain can be substituted with another C-beta branchingamino acid. Examples of C-beta branching amino acids include, e.g., I, Tand V.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/A binding domain. In an aspect of this embodiment, a BoNT/A bindingdomain comprises the binding domains of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/A binding domain comprises amino acids 874-1296 ofSEQ ID NO: 1. In another aspect of this embodiment, a BoNT/A bindingdomain comprises a naturally occurring BoNT/A binding domain variant,such as, e.g., an binding domain from a BoNT/A isoform or an bindingdomain from a BoNT/A subtype. In another aspect of this embodiment, aBoNT/A binding domain comprises a naturally occurring BoNT/A bindingdomain variant of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO: 5, such as, e.g., a BoNT/A isoform binding domain or aBoNT/A subtype binding domain. In another aspect of this embodiment, aBoNT/A binding domain comprises amino acids 874-1296 of a naturallyoccurring BoNT/A binding domain variant of SEQ ID NO: 1, such as, e.g.,a BoNT/A isoform binding domain or a BoNT/A subtype binding domain. Instill another aspect of this embodiment, a BoNT/A binding domaincomprises a non-naturally occurring BoNT/A binding domain variant, suchas, e.g., a conservative BoNT/A binding domain variant, anon-conservative BoNT/A binding domain variant, an active BoNT/A bindingdomain fragment, or any combination thereof. In still another aspect ofthis embodiment, a BoNT/A binding domain comprises the binding domain ofa non-naturally occurring BoNT/A binding domain variant of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, such as,e.g., a conservative BoNT/A binding domain variant, a non-conservativeBoNT/A binding domain variant, an active BoNT/A binding domain fragment,or any combination thereof. In still another aspect of this embodiment,a BoNT/A binding domain comprises amino acids 874-1296 of anon-naturally occurring BoNT/A binding domain variant of SEQ ID NO: 1,such as, e.g., a conservative BoNT/A binding domain variant, anon-conservative BoNT/A binding domain variant, an active BoNT/A bindingdomain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/A binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5; or at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, or at most 95% to the binding domain of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In yetother aspects of this embodiment, a BoNT/A binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 874-1296 of SEQ ID NO: 1; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to amino acids874-1296 of SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQID NO: 5. In yet other aspects of this embodiment, a BoNT/A bindingdomain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 874-1296 of SEQID NO: 1; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 874-1296 of SEQ ID NO: 1. In still other aspectsof this embodiment, a BoNT/A binding domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, or SEQ ID NO: 5; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the binding domain of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In further otheraspects of this embodiment, a BoNT/A binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 874-1296 of SEQ ID NO: 1; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 874-1296 of SEQ ID NO: 1.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/B binding domain. In an aspect of this embodiment, a BoNT/B bindingdomain comprises the binding domains of SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In other aspects of thisembodiment, a BoNT/B binding domain comprises amino acids 861-1291 ofSEQ ID NO: 6. In another aspect of this embodiment, a BoNT/B bindingdomain comprises a naturally occurring BoNT/B binding domain variant,such as, e.g., an binding domain from a BoNT/B isoform or an bindingdomain from a BoNT/B subtype. In another aspect of this embodiment, aBoNT/B binding domain comprises a naturally occurring BoNT/B bindingdomain variant of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ ID NO: 10, such as, e.g., a BoNT/B isoform binding domain or aBoNT/B subtype binding domain. In another aspect of this embodiment, aBoNT/B binding domain comprises amino acids 861-1291 of a naturallyoccurring BoNT/B binding domain variant of SEQ ID NO: 6, such as, e.g.,a BoNT/B isoform binding domain or a BoNT/B subtype binding domain. Instill another aspect of this embodiment, a BoNT/B binding domaincomprises a non-naturally occurring BoNT/B binding domain variant, suchas, e.g., a conservative BoNT/B binding domain variant, anon-conservative BoNT/B binding domain variant, an active BoNT/B bindingdomain fragment, or any combination thereof. In still another aspect ofthis embodiment, a BoNT/B binding domain comprises the binding domain ofa non-naturally occurring BoNT/B binding domain variant of SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, such as,e.g., a conservative BoNT/B binding domain variant, a non-conservativeBoNT/B binding domain variant, an active BoNT/B binding domain fragment,or any combination thereof. In still another aspect of this embodiment,a BoNT/B binding domain comprises amino acids 861-1291 of anon-naturally occurring BoNT/B binding domain variant of SEQ ID NO: 6,such as, e.g., a conservative BoNT/B binding domain variant, anon-conservative BoNT/B binding domain variant, an active BoNT/B bindingdomain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/B binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or SEQ ID NO: 10; or at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, or at most 95% to the binding domain of SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Inyet other aspects of this embodiment, a BoNT/B binding domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 861-1291 of SEQ ID NO: 6; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to amino acids861-1291 of SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/B binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQID NO: 10. In yet other aspects of this embodiment, a BoNT/B bindingdomain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 861-1291 of SEQID NO: 6; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 861-1291 of SEQ ID NO: 6. In still other aspectsof this embodiment, a BoNT/B binding domain comprises a polypeptidehaving, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, or SEQ ID NO: 10; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to the binding domain of SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In further otheraspects of this embodiment, a BoNT/B binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 861-1291 of SEQ ID NO: 6; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 861-1291 of SEQ ID NO: 6.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/C1 binding domain. In an aspect of this embodiment, a BoNT/C1binding domain comprises the binding domains of SEQ ID NO: 11 or SEQ IDNO: 12. In other aspects of this embodiment, a BoNT/C1 binding domaincomprises amino acids 869-1291 of SEQ ID NO: 11. In another aspect ofthis embodiment, a BoNT/C1 binding domain comprises a naturallyoccurring BoNT/C1 binding domain variant, such as, e.g., an bindingdomain from a BoNT/C1 isoform or an binding domain from a BoNT/C1subtype. In another aspect of this embodiment, a BoNT/C1 binding domaincomprises a naturally occurring BoNT/C1 binding domain variant of SEQ IDNO: 11 or SEQ ID NO: 12, such as, e.g., a BoNT/C1 isoform binding domainor a BoNT/C1 subtype binding domain. In another aspect of thisembodiment, a BoNT/C1 binding domain comprises amino acids 869-1291 of anaturally occurring BoNT/C1 binding domain variant of SEQ ID NO: 11,such as, e.g., a BoNT/C1 isoform binding domain or a BoNT/C1 subtypebinding domain. In still another aspect of this embodiment, a BoNT/C1binding domain comprises a non-naturally occurring BoNT/C1 bindingdomain variant, such as, e.g., a conservative BoNT/C1 binding domainvariant, a non-conservative BoNT/C1 binding domain variant, an activeBoNT/C1 binding domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/C1 binding domain comprisesthe binding domain of a non-naturally occurring BoNT/C1 binding domainvariant of SEQ ID NO: 11 or SEQ ID NO: 12, such as, e.g., a conservativeBoNT/C1 binding domain variant, a non-conservative BoNT/C1 bindingdomain variant, an active BoNT/C1 binding domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/C1 binding domain comprises amino acids 869-1291 of a non-naturallyoccurring BoNT/C1 binding domain variant of SEQ ID NO: 11, such as,e.g., a conservative BoNT/C1 binding domain variant, a non-conservativeBoNT/C1 binding domain variant, an active BoNT/C1 binding domainfragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/C1 binding domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 11 or SEQ ID NO: 12; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to thebinding domain of SEQ ID NO: 11 or SEQ ID NO: 12. In yet other aspectsof this embodiment, a BoNT/C1 binding domain comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to amino acids869-1291 of SEQ ID NO: 11; or at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, or at most 95% to amino acids 869-1291 of SEQ IDNO: 11.

In other aspects of this embodiment, a BoNT/C1 binding domain comprisesa polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the binding domain of SEQ ID NO: 11 orSEQ ID NO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 11 or SEQ IDNO: 12. In yet other aspects of this embodiment, a BoNT/C1 bindingdomain comprises a polypeptide having, e.g., at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 869-1291 of SEQID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 869-1291 of SEQ ID NO: 11. In still otheraspects of this embodiment, a BoNT/C1 binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 11 or SEQ IDNO: 12; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 11 or SEQ ID NO: 12. Infurther other aspects of this embodiment, a BoNT/C1 binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 869-1291 of SEQID NO: 11; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 869-1291 of SEQ ID NO: 11.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/D binding domain. In an aspect of this embodiment, a BoNT/D bindingdomain comprises the binding domains of SEQ ID NO: 13 or SEQ ID NO: 14.In other aspects of this embodiment, a BoNT/D binding domain comprisesamino acids 865-1291 of SEQ ID NO: 13. In another aspect of thisembodiment, a BoNT/D binding domain comprises a naturally occurringBoNT/D binding domain variant, such as, e.g., an binding domain from aBoNT/D isoform or an binding domain from a BoNT/D subtype. In anotheraspect of this embodiment, a BoNT/D binding domain comprises a naturallyoccurring BoNT/D binding domain variant of SEQ ID NO: 13 or SEQ ID NO:14, such as, e.g., a BoNT/D isoform binding domain or a BoNT/D subtypebinding domain. In another aspect of this embodiment, a BoNT/D bindingdomain comprises amino acids 865-1291 of a naturally occurring BoNT/Dbinding domain variant of SEQ ID NO: 13, such as, e.g., a BoNT/D isoformbinding domain or a BoNT/D subtype binding domain. In still anotheraspect of this embodiment, a BoNT/D binding domain comprises anon-naturally occurring BoNT/D binding domain variant, such as, e.g., aconservative BoNT/D binding domain variant, a non-conservative BoNT/Dbinding domain variant, an active BoNT/D binding domain fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D binding domain comprises the binding domain of a non-naturallyoccurring BoNT/D binding domain variant of SEQ ID NO: 13 or SEQ ID NO:14, such as, e.g., a conservative BoNT/D binding domain variant, anon-conservative BoNT/D binding domain variant, an active BoNT/D bindingdomain fragment, or any combination thereof. In still another aspect ofthis embodiment, a BoNT/D binding domain comprises amino acids 865-1291of a non-naturally occurring BoNT/D binding domain variant of SEQ ID NO:13, such as, e.g., a conservative BoNT/D binding domain variant, anon-conservative BoNT/D binding domain variant, an active BoNT/D bindingdomain fragment, or any combination thereof.

In other aspects of this embodiment, a BoNT/D binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 13 or SEQ ID NO: 14; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to thebinding domain of SEQ ID NO: 13 or SEQ ID NO: 14. In yet other aspectsof this embodiment, a BoNT/D binding domain comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to amino acids865-1291 of SEQ ID NO: 13; or at most 70%, at most 75%, at most 80%, atmost 85%, at most 90%, or at most 95% to amino acids 865-1291 of SEQ IDNO: 13.

In other aspects of this embodiment, a BoNT/D binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 13 or SEQ IDNO: 14; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 13 or SEQ ID NO: 14. In yetother aspects of this embodiment, a BoNT/D binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 865-1291 of SEQ ID NO: 13; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 865-1291 of SEQ ID NO: 13. In still otheraspects of this embodiment, a BoNT/D binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 13 or SEQ IDNO: 14; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 13 or SEQ ID NO: 14. Infurther other aspects of this embodiment, a BoNT/D binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 865-1291 of SEQID NO: 13; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 865-1291 of SEQ ID NO: 13.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/E binding domain. In an aspect of this embodiment, a BoNT/E bindingdomain comprises the binding domains of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17. In other aspects of this embodiment, a BoNT/E bindingdomain comprises amino acids 848-1252 of SEQ ID NO: 15. In anotheraspect of this embodiment, a BoNT/E binding domain comprises a naturallyoccurring BoNT/E binding domain variant, such as, e.g., an bindingdomain from a BoNT/E isoform or an binding domain from a BoNT/E subtype.In another aspect of this embodiment, a BoNT/E binding domain comprisesa naturally occurring BoNT/E binding domain variant of SEQ ID NO: 15,SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a BoNT/E isoform bindingdomain or a BoNT/E subtype binding domain. In another aspect of thisembodiment, a BoNT/E binding domain comprises amino acids 848-1252 of anaturally occurring BoNT/E binding domain variant of SEQ ID NO: 15, suchas, e.g., a BoNT/E isoform binding domain or a BoNT/E subtype bindingdomain. In still another aspect of this embodiment, a BoNT/E bindingdomain comprises a non-naturally occurring BoNT/E binding domainvariant, such as, e.g., a conservative BoNT/E binding domain variant, anon-conservative BoNT/E binding domain variant, an active BoNT/E bindingdomain fragment, or any combination thereof. In still another aspect ofthis embodiment, a BoNT/E binding domain comprises the binding domain ofa non-naturally occurring BoNT/E binding domain variant of SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO: 17, such as, e.g., a conservativeBoNT/E binding domain variant, a non-conservative BoNT/E binding domainvariant, an active BoNT/E binding domain fragment, or any combinationthereof. In still another aspect of this embodiment, a BoNT/E bindingdomain comprises amino acids 848-1252 of a non-naturally occurringBoNT/E binding domain variant of SEQ ID NO: 15, such as, e.g., aconservative BoNT/E binding domain variant, a non-conservative BoNT/Ebinding domain variant, an active BoNT/E binding domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BoNT/E binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; orat most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or atmost 95% to the binding domain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQID NO: 17. In yet other aspects of this embodiment, a BoNT/E bindingdomain comprises a polypeptide having an amino acid identity of, e.g.,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% to amino acids 848-1252 of SEQ ID NO: 15; or at most 70%,at most 75%, at most 80%, at most 85%, at most 90%, or at most 95% toamino acids 848-1252 of SEQ ID NO: 15.

In other aspects of this embodiment, a BoNT/E binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 15, SEQ IDNO: 16, or SEQ ID NO: 17; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the binding domain of SEQ ID NO: 15,SEQ ID NO: 16, or SEQ ID NO: 17. In yet other aspects of thisembodiment, a BoNT/E binding domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 848-1252 of SEQ ID NO: 15; or at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids848-1252 of SEQ ID NO: 15. In still other aspects of this embodiment, aBoNT/E binding domain comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In furtherother aspects of this embodiment, a BoNT/E binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 848-1252 of SEQ ID NO: 15; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 848-1252 of SEQ ID NO: 15.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/F binding domain. In an aspect of this embodiment, a BoNT/F bindingdomain comprises the binding domains of SEQ ID NO: 18, SEQ ID NO: 19, orSEQ ID NO: 20. In other aspects of this embodiment, a BoNT/F bindingdomain comprises amino acids 866-1274 of SEQ ID NO: 18. In anotheraspect of this embodiment, a BoNT/F binding domain comprises a naturallyoccurring BoNT/F binding domain variant, such as, e.g., an bindingdomain from a BoNT/F isoform or an binding domain from a BoNT/F subtype.In another aspect of this embodiment, a BoNT/F binding domain comprisesa naturally occurring BoNT/F binding domain variant of SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a BoNT/F isoform bindingdomain or a BoNT/F subtype binding domain. In another aspect of thisembodiment, a BoNT/F binding domain comprises amino acids 866-1274 of anaturally occurring BoNT/F binding domain variant of SEQ ID NO: 18, suchas, e.g., a BoNT/F isoform binding domain or a BoNT/F subtype bindingdomain. In still another aspect of this embodiment, a BoNT/F bindingdomain comprises a non-naturally occurring BoNT/F binding domainvariant, such as, e.g., a conservative BoNT/F binding domain variant, anon-conservative BoNT/F binding domain variant, an active BoNT/F bindingdomain fragment, or any combination thereof. In still another aspect ofthis embodiment, a BoNT/F binding domain comprises the binding domain ofa non-naturally occurring BoNT/F binding domain variant of SEQ ID NO:18, SEQ ID NO: 19, or SEQ ID NO: 20, such as, e.g., a conservativeBoNT/F binding domain variant, a non-conservative BoNT/F binding domainvariant, an active BoNT/F binding domain fragment, or any combinationthereof. In still another aspect of this embodiment, a BoNT/F bindingdomain comprises amino acids 866-1274 of a non-naturally occurringBoNT/F binding domain variant of SEQ ID NO: 18, such as, e.g., aconservative BoNT/F binding domain variant, a non-conservative BoNT/Fbinding domain variant, an active BoNT/F binding domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BoNT/F binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; orat most 70%, at most 75%, at most 80%, at most 85%, at most 90%, or atmost 95% to the binding domain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQID NO: 20. In yet other aspects of this embodiment, a BoNT/F bindingdomain comprises a polypeptide having an amino acid identity of, e.g.,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% to amino acids 866-1274 of SEQ ID NO: 18; or at most 70%,at most 75%, at most 80%, at most 85%, at most 90%, or at most 95% toamino acids 866-1274 of SEQ ID NO: 18.

In other aspects of this embodiment, a BoNT/F binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 18, SEQ IDNO: 19, or SEQ ID NO: 20; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 non-contiguous amino acid deletions, additions,and/or substitutions relative to the binding domain of SEQ ID NO: 18,SEQ ID NO: 19, or SEQ ID NO: 20. In yet other aspects of thisembodiment, a BoNT/F binding domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 866-1274 of SEQ ID NO: 18; or at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids866-1274 of SEQ ID NO: 18. In still other aspects of this embodiment, aBoNT/F binding domain comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. In furtherother aspects of this embodiment, a BoNT/F binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 866-1274 of SEQ ID NO: 18; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 866-1274 of SEQ ID NO: 18.

In another embodiment, a Clostridial toxin binding domain comprises aBoNT/G binding domain. In an aspect of this embodiment, a BoNT/G bindingdomain comprises the binding domains of SEQ ID NO: 21. In other aspectsof this embodiment, a BoNT/G binding domain comprises amino acids866-1297 of SEQ ID NO: 21. In another aspect of this embodiment, aBoNT/G binding domain comprises a naturally occurring BoNT/G bindingdomain variant, such as, e.g., an binding domain from a BoNT/G isoformor an binding domain from a BoNT/G subtype. In another aspect of thisembodiment, a BoNT/G binding domain comprises a naturally occurringBoNT/G binding domain variant of SEQ ID NO: 21, such as, e.g., a BoNT/Gisoform binding domain or a BoNT/G subtype binding domain. In anotheraspect of this embodiment, a BoNT/G binding domain comprises amino acids866-1297 of a naturally occurring BoNT/G binding domain variant of SEQID NO: 21, such as, e.g., a BoNT/G isoform binding domain or a BoNT/Gsubtype binding domain. In still another aspect of this embodiment, aBoNT/G binding domain comprises a non-naturally occurring BoNT/G bindingdomain variant, such as, e.g., a conservative BoNT/G binding domainvariant, a non-conservative BoNT/G binding domain variant, an activeBoNT/G binding domain fragment, or any combination thereof. In stillanother aspect of this embodiment, a BoNT/G binding domain comprises thebinding domain of a non-naturally occurring BoNT/G binding domainvariant of SEQ ID NO: 21, such as, e.g., a conservative BoNT/G bindingdomain variant, a non-conservative BoNT/G binding domain variant, anactive BoNT/G binding domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BoNT/G binding domaincomprises amino acids 866-1297 of a non-naturally occurring BoNT/Gbinding domain variant of SEQ ID NO: 21, such as, e.g., a conservativeBoNT/G binding domain variant, a non-conservative BoNT/G binding domainvariant, an active BoNT/G binding domain fragment, or any combinationthereof.

In other aspects of this embodiment, a BoNT/G binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 21; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the binding domainof SEQ ID NO: 21. In yet other aspects of this embodiment, a BoNT/Gbinding domain comprises a polypeptide having an amino acid identity of,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% to amino acids 866-1297 of SEQ ID NO: 21; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to amino acids 866-1297 of SEQ ID NO: 21.

In other aspects of this embodiment, a BoNT/G binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 21; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 21. In yet other aspects ofthis embodiment, a BoNT/G binding domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 866-1297 of SEQ ID NO: 21; or at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids866-1297 of SEQ ID NO: 21. In still other aspects of this embodiment, aBoNT/G binding domain comprises a polypeptide having, e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 21. Infurther other aspects of this embodiment, a BoNT/G binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 866-1297 of SEQID NO: 21; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 866-1297 of SEQ ID NO: 21.

In another embodiment, a Clostridial toxin binding domain comprises aTeNT binding domain. In an aspect of this embodiment, a TeNT bindingdomain comprises the binding domains of SEQ ID NO: 22. In other aspectsof this embodiment, a TeNT binding domain comprises amino acids 882-1315of SEQ ID NO: 22. In another aspect of this embodiment, a TeNT bindingdomain comprises a naturally occurring TeNT binding domain variant, suchas, e.g., an binding domain from a TeNT isoform or an binding domainfrom a TeNT subtype. In another aspect of this embodiment, a TeNTbinding domain comprises a naturally occurring TeNT binding domainvariant of SEQ ID NO: 22, such as, e.g., a TeNT isoform binding domainor a TeNT subtype binding domain. In another aspect of this embodiment,a TeNT binding domain comprises amino acids 882-1315 of a naturallyoccurring TeNT binding domain variant of SEQ ID NO: 22, such as, e.g., aTeNT isoform binding domain or a TeNT subtype binding domain. In stillanother aspect of this embodiment, a TeNT binding domain comprises anon-naturally occurring TeNT binding domain variant, such as, e.g., aconservative TeNT binding domain variant, a non-conservative TeNTbinding domain variant, an active TeNT binding domain fragment, or anycombination thereof. In still another aspect of this embodiment, a TeNTbinding domain comprises the binding domain of a non-naturally occurringTeNT binding domain variant of SEQ ID NO: 22, such as, e.g., aconservative TeNT binding domain variant, a non-conservative TeNTbinding domain variant, an active TeNT binding domain fragment, or anycombination thereof. In still another aspect of this embodiment, a TeNTbinding domain comprises amino acids 882-1315 of a non-naturallyoccurring TeNT binding domain variant of SEQ ID NO: 22, such as, e.g., aconservative TeNT binding domain variant, a non-conservative TeNTbinding domain variant, an active TeNT binding domain fragment, or anycombination thereof.

In other aspects of this embodiment, a TeNT binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 22; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the binding domainof SEQ ID NO: 22. In yet other aspects of this embodiment, a TeNTbinding domain comprises a polypeptide having an amino acid identity of,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% to amino acids 882-1315 of SEQ ID NO: 22; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to amino acids 882-1315 of SEQ ID NO: 22.

In other aspects of this embodiment, a TeNT binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 22; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 22. In yet other aspects ofthis embodiment, a TeNT binding domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 882-1315 of SEQ ID NO: 22; or at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids882-1315 of SEQ ID NO: 22.

In still other aspects of this embodiment, a TeNT binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to the binding domain of SEQ IDNO: 22; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 22. In further otheraspects of this embodiment, a TeNT binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 882-1315 of SEQ ID NO: 22; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguousamino acid deletions, additions, and/or substitutions relative to aminoacids 882-1315 of SEQ ID NO: 22.

In another embodiment, a Clostridial toxin binding domain comprises aBaNT binding domain. In an aspect of this embodiment, a BaNT bindingdomain comprises the binding domains of SEQ ID NO: 23. In other aspectsof this embodiment, a BaNT binding domain comprises amino acids 858-1268of SEQ ID NO: 23. In another aspect of this embodiment, a BaNT bindingdomain comprises a naturally occurring BaNT binding domain variant, suchas, e.g., an binding domain from a BaNT isoform or an binding domainfrom a BaNT subtype. In another aspect of this embodiment, a BaNTbinding domain comprises a naturally occurring BaNT binding domainvariant of SEQ ID NO: 23, such as, e.g., a BaNT isoform binding domainor a BaNT subtype binding domain. In another aspect of this embodiment,a BaNT binding domain comprises amino acids 858-1268 of a naturallyoccurring BaNT binding domain variant of SEQ ID NO: 23, such as, e.g., aBaNT isoform binding domain or a BaNT subtype binding domain. In stillanother aspect of this embodiment, a BaNT binding domain comprises anon-naturally occurring BaNT binding domain variant, such as, e.g., aconservative BaNT binding domain variant, a non-conservative BaNTbinding domain variant, an active BaNT binding domain fragment, or anycombination thereof. In still another aspect of this embodiment, a BaNTbinding domain comprises the binding domain of a non-naturally occurringBaNT binding domain variant of SEQ ID NO: 23, such as, e.g., aconservative BaNT binding domain variant, a non-conservative BaNTbinding domain variant, an active BaNT binding domain fragment, or anycombination thereof. In still another aspect of this embodiment, a BaNTbinding domain comprises amino acids 858-1268 of a non-naturallyoccurring BaNT binding domain variant of SEQ ID NO: 23, such as, e.g., aconservative BaNT binding domain variant, a non-conservative BaNTbinding domain variant, an active BaNT binding domain fragment, or anycombination thereof.

In other aspects of this embodiment, a BaNT binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 23; or at most 70%, at most 75%, atmost 80%, at most 85%, at most 90%, or at most 95% to the binding domainof SEQ ID NO: 23. In yet other aspects of this embodiment, a BaNTbinding domain comprises a polypeptide having an amino acid identity of,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% to amino acids 858-1268 of SEQ ID NO: 23; or atmost 70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most95% to amino acids 858-1268 of SEQ ID NO: 23.

In other aspects of this embodiment, a BaNT binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 23; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 23. In yet other aspects ofthis embodiment, a BaNT binding domain comprises a polypeptide having,e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 858-1268 of SEQ ID NO: 23; or at most 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 non-contiguous amino aciddeletions, additions, and/or substitutions relative to amino acids858-1268 of SEQ ID NO: 23. In still other aspects of this embodiment, aBaNT binding domain comprises a polypeptide having, e.g., at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 contiguous amino aciddeletions, additions, and/or substitutions relative to the bindingdomain of SEQ ID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 23. Infurther other aspects of this embodiment, a BaNT binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 858-1268 of SEQID NO: 23; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 858-1268 of SEQ ID NO: 23.

In another embodiment, a Clostridial toxin binding domain comprises aBuNT binding domain. In an aspect of this embodiment, a BuNT bindingdomain comprises the binding domains of SEQ ID NO: 24 or SEQ ID NO: 25.In other aspects of this embodiment, a BuNT binding domain comprisesamino acids 848-1251 of SEQ ID NO: 24. In another aspect of thisembodiment, a BuNT binding domain comprises a naturally occurring BuNTbinding domain variant, such as, e.g., an binding domain from a BuNTisoform or an binding domain from a BuNT subtype. In another aspect ofthis embodiment, a BuNT binding domain comprises a naturally occurringBuNT binding domain variant of SEQ ID NO: 24 or SEQ ID NO: 25, such as,e.g., a BuNT isoform binding domain or a BuNT subtype binding domain. Inanother aspect of this embodiment, a BuNT binding domain comprises aminoacids 848-1251 of a naturally occurring BuNT binding domain variant ofSEQ ID NO: 24, such as, e.g., a BuNT isoform binding domain or a BuNTsubtype binding domain. In still another aspect of this embodiment, aBuNT binding domain comprises a non-naturally occurring BuNT bindingdomain variant, such as, e.g., a conservative BuNT binding domainvariant, a non-conservative BuNT binding domain variant, an active BuNTbinding domain fragment, or any combination thereof. In still anotheraspect of this embodiment, a BuNT binding domain comprises the bindingdomain of a non-naturally occurring BuNT binding domain variant of SEQID NO: 24 or SEQ ID NO: 25, such as, e.g., a conservative BuNT bindingdomain variant, a non-conservative BuNT binding domain variant, anactive BuNT binding domain fragment, or any combination thereof. Instill another aspect of this embodiment, a BuNT binding domain comprisesamino acids 848-1251 of a non-naturally occurring BuNT binding domainvariant of SEQ ID NO: 24, such as, e.g., a conservative BuNT bindingdomain variant, a non-conservative BuNT binding domain variant, anactive BuNT binding domain fragment, or any combination thereof.

In other aspects of this embodiment, a BuNT binding domain comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% tothe binding domain of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to thebinding domain of SEQ ID NO: 24 or SEQ ID NO: 25. In yet other aspectsof this embodiment, a BuNT binding domain comprises a polypeptide havingan amino acid identity of, e.g., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% to amino acids 848-1251of SEQ ID NO: 24 or SEQ ID NO: 25; or at most 70%, at most 75%, at most80%, at most 85%, at most 90%, or at most 95% to amino acids 848-1251 ofSEQ ID NO: 24 or SEQ ID NO: 25.

In other aspects of this embodiment, a BuNT binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 24 or SEQ IDNO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100non-contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 240R SEQ ID NO: 25. In yetother aspects of this embodiment, a BuNT binding domain comprises apolypeptide having, e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, or 100 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to amino acids 848-1251 of SEQ ID NO: 24 or SEQID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or100 non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 848-1251 of SEQ ID NO: 24 or SEQ ID NO: 25. Instill other aspects of this embodiment, a BuNT binding domain comprisesa polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, or 100 contiguous amino acid deletions, additions, and/orsubstitutions relative to the binding domain of SEQ ID NO: 24 or SEQ IDNO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100contiguous amino acid deletions, additions, and/or substitutionsrelative to the binding domain of SEQ ID NO: 24 or SEQ ID NO: 25. Infurther other aspects of this embodiment, a BuNT binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, or 100 contiguous amino acid deletions,additions, and/or substitutions relative to amino acids 848-1251 of SEQID NO: 24 or SEQ ID NO: 25; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, or 100 contiguous amino acid deletions, additions,and/or substitutions relative to amino acids 848-1251 of SEQ ID NO: 24or SEQ ID NO: 25.

Aspects of the present specification provide, in part, a non-Clostridialtoxin binding domain. As used herein, the term “non-Clostridial toxinbinding domain” refers to any polypeptide that can execute the bindingstep of the intoxication process that initiates the overallinternalization mechanism whereby the modified Clostridial toxindisclosed in the present specification intoxicates a target cell.Examples of binding domains are described in, e.g., Keith A. Foster etal., Clostridial Toxin Derivatives Able To Modify Peripheral SensoryAfferent Functions, U.S. Pat. No. 5,989,545; Clifford C. Shone et al.,Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617; Conrad P. Quinn etal., Methods and Compounds for the Treatment of Mucus Hypersecretion,U.S. Pat. No. 6,632,440; Lance E. Steward et al., Methods AndCompositions For The Treatment Of Pancreatitis, U.S. Pat. No. 6,843,998;J. Oliver Dolly et al., Activatable Recombinant Neurotoxins, U.S. Pat.No. 7,132,259; Stephan Donovan, Clostridial Toxin Derivatives andMethods For Treating Pain, U.S. Patent Publication 2002/0037833; KeithA. Foster et al., Inhibition of Secretion from Non-neural Cells, U.S.Patent Publication 2003/0180289; Lance E. Steward et al., MultivalentClostridial Toxin Derivatives and Methods of Their Use, U.S. PatentPublication 2006/0211619; Keith A. Foster et al., Non-Cytotoxic ProteinConjugates, U.S. Patent Publication 2008/0187960; Steward, L. E. et al.,Modified Clostridial Toxins with Enhanced Translocation Capabilities andAltered Targeting Activity For Non-Clostridial Toxin Target Cells, U.S.patent application Ser. No. 11/776,075; Keith A. Foster et al.,Re-targeted Toxin Conjugates, U.S. patent application Ser. No.11/792,210; each of which is incorporated by reference in its entirety.

A non-Clostridial toxin binding domain includes, without limitation,naturally occurring non-Clostridial toxin binding domain variants, suchas, e.g., non-Clostridial toxin binding domain isoforms andnon-Clostridial toxin binding domain subtypes; and non-naturallyoccurring non-Clostridial toxin binding domain variants, such as, e.g.,conservative non-Clostridial toxin binding domain variants,non-conservative non-Clostridial toxin binding domain variants,non-Clostridial toxin binding domain chimeras, active non-Clostridialtoxin binding domain fragments thereof, or any combination thereof.

As used herein, the term “non-Clostridial toxin binding domain variant,”whether naturally-occurring or non-naturally-occurring, refers to anon-Clostridial toxin binding domain that has at least one amino acidchange from the corresponding region of a reference sequence and can bedescribed in percent identity to the corresponding region of thatreference sequence. Unless expressly indicated, non-Clostridial toxinbinding domain variants useful to practice the disclosed embodiments arevariants that execute the binding step of the intoxication process.

It is recognized by those of skill in the art that within eachnon-Clostridial toxin binding domain there can be naturally occurringvariants that differ somewhat in their amino acid sequence, and also inthe nucleic acids encoding these proteins. As used herein, the term“naturally occurring non-Clostridial toxin binding domain variant”refers to any non-Clostridial toxin binding domain produced by anaturally-occurring process, including, without limitation,non-Clostridial toxin binding domain isoforms produced fromalternatively-spliced transcripts and non-Clostridial toxin bindingdomain isoforms produced by spontaneous mutation. A naturally occurringnon-Clostridial toxin binding domain variant can function insubstantially the same manner as the reference non-Clostridial toxinbinding domain on which the naturally occurring non-Clostridial toxinbinding domain variant is based, and can be substituted for thereference non-Clostridial toxin binding domain in any aspect of thepresent specification. A non-limiting examples of a naturally occurringnon-Clostridial toxin binding domain variant is a non-Clostridial toxinbinding domain isoform. Non-limiting examples of a non-Clostridial toxinbinding domain isoform include, e.g., opioid binding domain isoforms,tachykinin binding domain isoforms, melanocortin binding domainisoforms, galanin binding domain isoforms, granin binding domainisoforms, Neuropeptide Y related peptide binding domain isoforms,neurohormone binding domain isoforms, neuroregulatory cytokine bindingdomain isoforms, kinin peptide binding domain isoforms, growth factorbinding domain isoforms, and glucagon like hormone binding domainisoforms.

As used herein, the term “non-naturally occurring non-Clostridial toxinbinding domain variant” refers to any non-Clostridial toxin bindingdomain produced with the aid of human manipulation, including, withoutlimitation, non-Clostridial toxin binding domains produced by geneticengineering using random mutagenesis or rational design andnon-Clostridial toxin binding domains produced by chemical synthesis.Non-limiting examples of non-naturally occurring non-Clostridial toxinbinding domain variants include, e.g., conservative non-Clostridialtoxin binding domain variants, non-conservative non-Clostridial toxinbinding domain variants, non-Clostridial toxin binding domain chimericvariants and active non-Clostridial toxin binding domain fragments.

As used herein, the term “conservative non-Clostridial toxin bindingdomain variant” refers to a non-Clostridial toxin binding domain thathas at least one amino acid substituted by another amino acid or anamino acid analog that has at least one property similar to that of theoriginal amino acid from a reference non-Clostridial toxin bindingdomain sequence. Examples of properties include, without limitation,similar size, topography, charge, hydrophobicity, hydrophilicity,lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, aphysicochemical property, of the like, or any combination thereof. Aconservative non-Clostridial toxin binding domain variant can functionin substantially the same manner as the reference non-Clostridial toxinbinding domain on which the conservative non-Clostridial toxin bindingdomain variant is based, and can be substituted for the referencenon-Clostridial toxin binding domain in any aspect of the presentspecification. Non-limiting examples of a conservative non-Clostridialtoxin binding domain variant include, e.g., conservative opioid bindingdomain variants, conservative tachykinin binding domain variants,conservative melanocortin binding domain variants, conservative galaninbinding domain variants, conservative granin binding domain variants,conservative Neuropeptide Y related peptide binding domain variants,conservative neurohormone binding domain variants, conservativeneuroregulatory cytokine binding domain variants, conservative kininpeptide binding domain variants, conservative growth factor bindingdomain variants, and conservative glucagon like hormone binding domainvariants.

As used herein, the term “non-conservative non-Clostridial toxin bindingdomain variant” refers to a non-Clostridial toxin binding domain inwhich 1) at least one amino acid is deleted from the referencenon-Clostridial toxin binding domain on which the non-conservativenon-Clostridial toxin binding domain variant is based; 2) at least oneamino acid added to the reference non-Clostridial toxin binding domainon which the non-conservative non-Clostridial toxin binding domain isbased; or 3) at least one amino acid is substituted by another aminoacid or an amino acid analog that does not share any property similar tothat of the original amino acid from a reference non-Clostridial toxinbinding domain sequence. A non-conservative non-Clostridial toxinbinding domain variant can function in substantially the same manner asthe reference non-Clostridial toxin binding domain on which thenon-conservative non-Clostridial toxin binding domain variant is based,and can be substituted for the reference non-Clostridial toxin bindingdomain in any aspect of the present specification. Non-limiting examplesof a non-conservative non-Clostridial toxin binding domain variantinclude, e.g., non-conservative opioid binding domain variants,non-conservative tachykinin binding domain variants, non-conservativemelanocortin binding domain variants, non-conservative galanin bindingdomain variants, non-conservative granin binding domain variants,non-conservative Neuropeptide Y related peptide binding domain variants,non-conservative neurohormone binding domain variants, non-conservativeneuroregulatory cytokine binding domain variants, non-conservative kininpeptide binding domain variants, non-conservative growth factor bindingdomain variants, and non-conservative glucagon like hormone bindingdomain variants.

As used herein, the term “active non-Clostridial toxin binding domainfragment” refers to any of a variety of Clostridial toxin fragmentscomprising the binding domain can be useful in aspects of the presentspecification with the proviso that these biding domain fragments canpreferentially interact with the cognate receptor, and thus participatein executing the overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate.

Any of a variety of sequence alignment methods can be used to determinepercent identity of naturally-occurring Clostridial toxin binding domainvariants and non-naturally-occurring Clostridial toxin binding domainvariants, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification comprises a non-Clostridial toxin binding domain.In an aspect of this embodiment, a non-Clostridial toxin binding domaincomprises a naturally occurring non-Clostridial toxin binding domainvariant, such as, e.g., a non-Clostridial toxin binding domain isoform.In another aspect of this embodiment, a non-Clostridial toxin bindingdomain comprises a non-naturally occurring non-Clostridial toxin bindingdomain variant, such as, e.g., a conservative non-Clostridial toxinbinding domain variant, a non-conservative non-Clostridial toxin bindingdomain variant, an active non-Clostridial toxin binding domain fragment,or any combination thereof.

In another embodiment, a hydrophobic amino acid at one particularposition in the polypeptide chain of the non-Clostridial toxin bindingdomain can be substituted with another hydrophobic amino acid. Examplesof hydrophobic amino acids include, e.g., C, F, I, L, M, V and W. Inanother aspect of this embodiment, an aliphatic amino acid at oneparticular position in the polypeptide chain of the non-Clostridialtoxin binding domain can be substituted with another aliphatic aminoacid. Examples of aliphatic amino acids include, e.g., A, I, L, P, andV. In yet another aspect of this embodiment, an aromatic amino acid atone particular position in the polypeptide chain of the non-Clostridialtoxin binding domain can be substituted with another aromatic aminoacid. Examples of aromatic amino acids include, e.g., F, H, W and Y. Instill another aspect of this embodiment, a stacking amino acid at oneparticular position in the polypeptide chain of the non-Clostridialtoxin binding domain can be substituted with another stacking aminoacid. Examples of stacking amino acids include, e.g., F, H, W and Y. Ina further aspect of this embodiment, a polar amino acid at oneparticular position in the polypeptide chain of the non-Clostridialtoxin binding domain can be substituted with another polar amino acid.Examples of polar amino acids include, e.g., D, E, K, N, Q, and R. In afurther aspect of this embodiment, a less polar or indifferent aminoacid at one particular position in the polypeptide chain of thenon-Clostridial toxin binding domain can be substituted with anotherless polar or indifferent amino acid. Examples of less polar orindifferent amino acids include, e.g., A, H, G, P, S, T, and Y. In a yetfurther aspect of this embodiment, a positive charged amino acid at oneparticular position in the polypeptide chain of the non-Clostridialtoxin binding domain can be substituted with another positive chargedamino acid. Examples of positive charged amino acids include, e.g., K,R, and H. In a still further aspect of this embodiment, a negativecharged amino acid at one particular position in the polypeptide chainof the non-Clostridial toxin binding domain can be substituted withanother negative charged amino acid. Examples of negative charged aminoacids include, e.g., D and E. In another aspect of this embodiment, asmall amino acid at one particular position in the polypeptide chain ofthe non-Clostridial toxin binding domain can be substituted with anothersmall amino acid. Examples of small amino acids include, e.g., A, D, G,N, P, S, and T. In yet another aspect of this embodiment, a C-betabranching amino acid at one particular position in the polypeptide chainof the non-Clostridial toxin binding domain can be substituted withanother C-beta branching amino acid. Examples of C-beta branching aminoacids include, e.g., I, T and V.

In another embodiment, a non-Clostridial toxin binding domain comprisesan opioid binding domain, such as, e.g., an enkephalin, an endomorphin,an endorphin, a dynorphin, a nociceptin or a hemorphin. In yet anotheraspect of this embodiment, a non-Clostridial toxin binding domaincomprises a tachykinin binding domain, such as, e.g., a Substance P, aneuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A(NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B(NKB), a hemokinin or a endokinin. In still another aspect of thisembodiment, a non-Clostridial toxin comprises a melanocortin bindingdomain, such as, e.g., a melanocyte stimulating hormone,adrenocorticotropin, or a lipotropin. In still another aspect of thisembodiment, a non-Clostridial toxin binding domain comprises a galaninbinding domain, such as, e.g., a galanin or a galanin message-associatedpeptide. In a further aspect of this embodiment, a non-Clostridial toxinbinding domain comprises a granin binding domain, such as, e.g., aChromogranin A, a Chromogranin B, or a Chromogranin C. In another aspectof this embodiment, a non-Clostridial toxin binding domain comprises aNeuropeptide Y related peptide binding domain, such as, e.g., aNeuropeptide Y, a Peptide YY, Pancreatic peptide or a Pancreaticicosapeptide. In yet another aspect of this embodiment, anon-Clostridial toxin binding domain comprises a neurohormone bindingdomain, such as, e.g., a corticotropin-releasing hormone, a parathyroidhormone, a thyrotropin-releasing hormone, or a somatostatin. In stillanother aspect of this embodiment, a non-Clostridial toxin bindingdomain comprises a neuroregulatory cytokine binding domain, such as,e.g., a ciliary neurotrophic factor, a glycophorin-A, a leukemiainhibitory factor, a cholinergic differentiation factor, an interleukin,an oncostatin M, a cardiotrophin-1, a cardiotrophin-like cytokine, or aneuroleukin. In a further aspect of this embodiment, a non-Clostridialtoxin binding domain comprises a kinin peptide binding domain, such as,e.g., a bradykinin, a kallidin, a desArg9 bradykinin, or a desArg10bradykinin. In another aspect of this embodiment, a non-Clostridialtoxin binding domain comprises a growth factor binding domain, such as,e.g., a fibroblast growth factor binding domain, a nerve growth factorbinding domain, an insulin growth factor binding domain, an epidermalgrowth factor binding domain, a vascular endothelial growth factorbinding domain, a brain derived neurotrophic factor binding domain, agrowth derived neurotrophic factor binding domain, a neurotrophinbinding domain, such as, e.g., a neurotrophin-3, a neurotrophin-4/5, ahead activator peptide binding domain, a neurturin binding domain, apersephin binding domain, an artemin binding domain, a transformationgrowth factor β binding domain, such as, e.g., a TGFβ1, a TGFβ2, a TGFβ3or a TGFβ4, a bone morphogenic protein binding domain, such as, e.g., aBMP2, a BMP3, a BMP4, a BMP5, a BMP6, a BMP7, a BMP8 or a BMP10, agrowth differentiation factor binding domain, such as, e.g., a GDF1, aGDF2, a GDF3, a GDF5, a GDF6, a GDF7, a GDF8, a GDF10, a GDF11 or aGDF15, or an activin binding domain, such as, e.g., an activin A, anactivin B, an activin C, an activin E or an inhibin A. In another aspectof this embodiment, a non-Clostridial toxin binding domain comprises aglucagon like hormone binding domain, such as, e.g., a secretin, aglucagon-like peptide, like a GLP-1 and a GLP-2, a pituitary adenylatecyclase activating peptide binding domain, a growth hormone-releasinghormone binding domain, vasoactive intestinal peptide binding domainlike a VIP1 or a VIP2, a gastric inhibitory polypeptide binding domain,a calcitonin-related peptide, visceral gut peptide binding domain like agastrin, a gastrin-releasing peptide or a cholecystokinin, or a PARpeptide binding domain like a PAR1 peptide, a PAR2 peptide, a PAR3peptide or a PAR4 peptide.

In another embodiment, an opioid peptide comprises an enkephalinpeptide. In aspects of this embodiment, an enkephalin peptide comprisesa Leu-enkephalin, a Met-enkephalin, a Met-enkephalin MRGL or aMet-enkephalin MRF. In other aspects of this embodiment, an enkephalinpeptide comprises SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ IDNO: 29.

In other aspects of this embodiment, an enkephalin comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toSEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29; or at most70%, at most 75%, at most 80%, at most 85%, at most 90%, or at most 95%to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. In yetother aspects of this embodiment, an enkephalin comprises a polypeptidehaving, e.g., at least 1, 2, or 3 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28 or SEQ ID NO: 29; or at most 1, 2, or 3 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. In still otheraspects of this embodiment, an enkephalin comprises a polypeptidehaving, e.g., at least 1, 2, or 3 contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28 or SEQ ID NO: 29; or at most 1, 2, or 3 contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.

In another embodiment, an opioid peptide comprises a bovineadrenomedullary-22 (BAM22) peptide. In aspects of this embodiment, aBAM22 peptide comprises a BAM22 peptide (1-12), a BAM22 peptide (6-22),a BAM22 peptide (8-22) or a BAM22 peptide (1-22). In other aspects ofthis embodiment, a BAM22 peptide comprises amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 30; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 31; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 32; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 33; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 34 oramino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 35.

In other aspects of this embodiment, a BAM22 peptide comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toamino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 30; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 31; amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 32; amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 33; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 34 or amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 35; or at most 70%, at most75%, at most 80%, at most 85%, at most 90%, or at most 95% to aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 30; amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 31; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 32; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 33;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 34 or amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 35.

In yet other aspects of this embodiment, a BAM22 peptide comprises apolypeptide having, e.g., at least 1, 2, 3, 4, or 5 non-contiguous aminoacid deletions, additions, and/or substitutions relative to amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 30; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 31; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 32; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 33; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 34 or amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 35; or at most 1, 2, 3, 4, or 5non-contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 30; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 31; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 32;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 33; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 34 or amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 35.

In still other aspects of this embodiment, a BAM22 peptide comprises apolypeptide having, e.g., at least 1, 2, 3, 4, or 5 contiguous aminoacid deletions, additions, and/or substitutions relative to amino acids1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ IDNO: 30; amino acids 1-12, amino acids 6-22, amino acids 8-22 or aminoacids 1-22 of SEQ ID NO: 31; amino acids 1-12, amino acids 6-22, aminoacids 8-22 or amino acids 1-22 of SEQ ID NO: 32; amino acids 1-12, aminoacids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 33; aminoacids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 ofSEQ ID NO: 34 or amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 35; or at most 1, 2, 3, 4, or 5contiguous amino acid deletions, additions, and/or substitutionsrelative to amino acids 1-12, amino acids 6-22, amino acids 8-22 oramino acids 1-22 of SEQ ID NO: 30; amino acids 1-12, amino acids 6-22,amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 71; amino acids 1-12,amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 32;amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1-22of SEQ ID NO: 33; amino acids 1-12, amino acids 6-22, amino acids 8-22or amino acids 1-22 of SEQ ID NO: 34 or amino acids 1-12, amino acids6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 35.

In another embodiment, an opioid peptide comprises an endomorphinpeptide. In aspects of this embodiment, an endomorphin peptide comprisesan endomorphin-1 or an endomorphin-2. In other aspects of thisembodiment, an endomorphin peptide comprises SEQ ID NO: 36 or SEQ ID NO:37.

In other aspects of this embodiment, an endomorphin comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toSEQ ID NO: 36 or SEQ ID NO: 37; or at most 70%, at most 75%, at most80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO: 36 or SEQ IDNO: 37. In yet other aspects of this embodiment, an endomorphincomprises a polypeptide having, e.g., at least 1, 2, or 3 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 36 or SEQ ID NO: 37; or at most 1, 2, or 3 non-contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 36 orSEQ ID NO: 37. In still other aspects of this embodiment, an endomorphincomprises a polypeptide having, e.g., at least 1, 2, or 3 contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 36 or SEQ ID NO: 37; or at most 1, 2, or 3 contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 36 orSEQ ID NO: 37.

In another embodiment, an opioid peptide comprises an endorphin peptide.In aspects of this embodiment, an endorphin peptide comprises anendorphin-α, a neoendorphin-α, an endorphin-β, a neoendorphin-β or anendorphin-γ. In other aspects of this embodiment, an endorphin peptidecomprises SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,SEQ ID NO: 42, or SEQ ID NO: 43. In other aspects of this embodiment, anendorphin comprises a polypeptide having an amino acid identity of,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43; or at most 70%, at most 75%,at most 80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ IDNO: 43. In yet other aspects of this embodiment, an endorphin comprisesa polypeptide having, e.g., at least 1, 2, 3, 4, or 5 non-contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, orSEQ ID NO: 43; or at most 1, 2, 3, 4, or 5 non-contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ IDNO: 43. In still other aspects of this embodiment, an endorphincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,SEQ ID NO: 42, or SEQ ID NO: 43; or at most 1, 2, 3, 4, or 5 contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, orSEQ ID NO: 43.

In another embodiment, an opioid peptide comprises a dynorphin peptide.In aspects of this embodiment, a dynorphin peptide comprises a dynorphinA, a dynorphin B (leumorphin) or a rimorphin. In other aspects of thisembodiment, a dynorphin peptide comprises SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQID NO: 74.

In other aspects of this embodiment, a dynorphin comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 44,SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ IDNO: 69; or at most 70%, at most 75%, at most 80%, at most 85%, at most90%, or at most 95% to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 69. In yet other aspects of thisembodiment, a dynorphin comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 44, SEQ ID NO:45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 69; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 44,SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ IDNO: 69. In still other aspects of this embodiment, a dynorphin comprisesa polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,SEQ ID NO: 53, or SEQ ID NO: 69; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,SEQ ID NO: 53, or SEQ ID NO: 69.

In another embodiment, an opioid peptide comprises a nociceptin peptide.In aspects of this embodiment, a nociceptin peptide comprises anociceptin RK, a nociceptin, a neuropeptide 1, a neuropeptide 2, or aneuropeptide 3. In other aspects of this embodiment, a nociceptinpeptide comprises SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQID NO: 83, or SEQ ID NO: 84.

In other aspects of this embodiment, a nociceptin comprises apolypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toSEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQID NO: 84; or at most 70%, at most 75%, at most 80%, at most 85%, atmost 90%, or at most 95% to SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:82, SEQ ID NO: 83, or SEQ ID NO: 84. In yet other aspects of thisembodiment, a nociceptin comprises a polypeptide having, e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84; or at most 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84. In still otheraspects of this embodiment, a nociceptin comprises a polypeptide having,e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino aciddeletions, additions, and/or substitutions relative to SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84; or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84.

In another embodiment, an opioid peptide comprises a hemorphin peptide.In aspects of this embodiment, a hemorphin peptide comprises a LVVH7, aVVH7, a VH7, a H7, a LVVH6, a LVVH5, VVH5, a LVVH4, and a LVVH3. Inother aspects of this embodiment, a hemorphin peptide comprises SEQ IDNO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93.

In other aspects of this embodiment, a hemorphin comprises a polypeptidehaving an amino acid identity of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% to SEQ ID NO: 85,SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93; or at most 70%, atmost 75%, at most 80%, at most 85%, at most 90%, or at most 95% to SEQID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93. In yetother aspects of this embodiment, a nociceptin comprises a polypeptidehaving, e.g., at least 1, 2, or 3 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 85, SEQ ID NO:86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ IDNO: 91, SEQ ID NO: 92, or SEQ ID NO: 93; or at most 1, 2, or 3non-contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88,SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ IDNO: 93. In still other aspects of this embodiment, a nociceptincomprises a polypeptide having, e.g., at least 1, 2, or 3 contiguousamino acid deletions, additions, and/or substitutions relative to SEQ IDNO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93; or at most 1,2, or 3 contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88,SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ IDNO: 93.

In yet another embodiment, a non-Clostridial toxin binding domaincomprises a galanin peptide binding domain. In aspects of thisembodiment, a galanin peptide binding domain comprises a galanin or agalanin message-associated peptide (GMAP). In other aspects of thisembodiment, a galanin peptide binding domain comprises SEQ ID NO: 94 orSEQ ID NO: 95.

In other aspects of this embodiment, a galanin binding domain comprisesa polypeptide having an amino acid identity of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% toSEQ ID NO: 94 or SEQ ID NO: 95; or at most 70%, at most 75%, at most80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO: 94 or SEQ IDNO: 95. In yet other aspects of this embodiment, a galanin bindingdomain comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 94 or SEQ ID NO: 95; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 non-contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 94 or SEQ ID NO:95. In still other aspects of this embodiment, a galanin binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 94 or SEQ ID NO: 95; or at most 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid deletions,additions, and/or substitutions relative to SEQ ID NO: 94 or SEQ ID NO:95.

In still another embodiment, a non-Clostridial toxin binding domaincomprises a tachykinin peptide binding domain. In aspects of thisembodiment, a tachykinin peptide binding domain comprises a Substance P,a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A(NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B(NKB), a hemokinin or a endokinin. In other aspects of this embodiment,a tachykinin peptide binding domain comprises SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,or SEQ ID NO: 107.

In other aspects of this embodiment, a tachykinin peptide binding domaincomprises a polypeptide having an amino acid identity of, e.g., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107; or at most 70%,at most 75%, at most 80%, at most 85%, at most 90%, or at most 95% toSEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107. In yet other aspects ofthis embodiment, a tachykinin peptide binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, or 5 non-contiguous aminoacid deletions, additions, and/or substitutions relative to SEQ ID NO:96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ IDNO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105,SEQ ID NO: 106, or SEQ ID NO: 107; or at most 1, 2, 3, 4, or 5non-contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ IDNO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107. In stillother aspects of this embodiment, a tachykinin peptide binding domaincomprises a polypeptide having, e.g., at least 1, 2, 3, 4, or 5contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ IDNO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107; or at most1, 2, 3, 4, or 5 contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ IDNO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO:107.

In still another embodiment, a non-Clostridial toxin binding domaincomprises a Neuropeptide Y related peptide binding domain. In aspects ofthis embodiment, a Neuropeptide Y related peptide binding domaincomprises a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide(PP) or a Pancreatic icosapeptide (PIP). In other aspects of thisembodiment, a Neuropeptide Y related peptide binding domain comprisesSEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQID NO: 112.

In other aspects of this embodiment, a Neuropeptide Y related peptidebinding domain comprises a polypeptide having an amino acid identity of,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,SEQ ID NO: 111, or SEQ ID NO: 112; or at most 70%, at most 75%, at most80%, at most 85%, at most 90%, or at most 95% to SEQ ID NO: 108, SEQ IDNO: 109, SEQ ID NO: 110, SEQ ID NO: 111, or SEQ ID NO: 112. In yet otheraspects of this embodiment, a Neuropeptide Y related peptide bindingdomain comprises a polypeptide having, e.g., at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, or SEQ ID NO: 112; or at most 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 non-contiguous amino acid deletions, additions, and/orsubstitutions relative to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID NO: 111, or SEQ ID NO: 112. In still other aspects of thisembodiment, a Neuropeptide Y related peptide binding domain comprises apolypeptide having, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, or SEQ ID NO: 112; or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10contiguous amino acid deletions, additions, and/or substitutionsrelative to SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, or SEQ ID NO: 112.

It is envisioned that a Clostridial toxin chimeric disclosed in thepresent specification can comprise a non-Clostridial binding domain inany and all locations with the proviso that Clostridial toxin chimericcan perform the intoxication process. Non-limiting examples include,locating a non-Clostridial binding domain at the amino terminus of amodified Clostridial toxin; locating a non-Clostridial binding domainbetween a Clostridial toxin enzymatic domain and a translocation domainof a modified Clostridial toxin; and locating a non-Clostridial bindingdomain at the carboxyl terminus of a modified Clostridial toxin. Othernon-limiting examples include, locating a non-Clostridial binding domainbetween a Clostridial toxin enzymatic domain and a Clostridial toxintranslocation domain of a modified Clostridial toxin. The enzymaticdomain of naturally-occurring Clostridial toxins contains the nativestart methionine. Thus, in domain organizations where the enzymaticdomain is not in the amino-terminal location an amino acid sequencecomprising the start methionine should be placed in front of theamino-terminal domain. Likewise, where a non-Clostridial binding domainis in the amino-terminal position, an amino acid sequence comprising astart methionine and a protease cleavage site may be operably-linked insituations in which a non-Clostridial binding domain requires a freeamino terminus, see, e.g., Shengwen Li et al., Degradable ClostridialToxins, U.S. patent application Ser. No. 11/572,512 (Jan. 23, 2007),which is hereby incorporated by reference in its entirety. In addition,it is known in the art that when adding a polypeptide that isoperably-linked to the amino terminus of another polypeptide comprisingthe start methionine that the original methionine residue can bedeleted.

Thus, in an embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising anon-Clostridial binding domain, a translocation domain, an exogenousprotease cleavage site and an enzymatic domain (FIG. 3A). In an aspectof this embodiment, a modified Clostridial toxin can comprise an aminoto carboxyl single polypeptide linear order comprising a non-Clostridialbinding domain, a Clostridial toxin translocation domain, an exogenousprotease cleavage site and a Clostridial toxin enzymatic domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising anon-Clostridial binding domain, an enzymatic domain, an exogenousprotease cleavage site, and a translocation domain (FIG. 3B). In anaspect of this embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising anon-Clostridial binding domain, a Clostridial toxin enzymatic domain, anexogenous protease cleavage site, a Clostridial toxin translocationdomain.

In yet another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising anenzymatic domain, an exogenous protease cleavage site, a non-Clostridialbinding domain, and a translocation domain (FIG. 4A). In an aspect ofthis embodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, an exogenous protease cleavage site, a non-Clostridialbinding domain, and a Clostridial toxin translocation domain.

In yet another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising atranslocation domain, an exogenous protease cleavage site, anon-Clostridial binding domain, and an enzymatic domain (FIG. 4B). In anaspect of this embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin translocation domain, a non-Clostridial bindingdomain, an exogenous protease cleavage site and a Clostridial toxinenzymatic domain.

In another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising anenzymatic domain, a non-Clostridial binding domain, an exogenousprotease cleavage site, and a translocation domain (FIG. 4C). In anaspect of this embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising aClostridial toxin enzymatic domain, a non-Clostridial binding domain, anexogenous protease cleavage site, a Clostridial toxin translocationdomain.

In yet another embodiment, a modified Clostridial toxin can comprise anamino to carboxyl single polypeptide linear order comprising atranslocation domain, a non-Clostridial binding domain, an exogenousprotease cleavage site and an enzymatic domain (FIG. 4D). In an aspectof this embodiment, a modified Clostridial toxin can comprise an aminoto carboxyl single polypeptide linear order comprising a Clostridialtoxin translocation domain, a non-Clostridial binding domain, anexogenous protease cleavage site and a Clostridial toxin enzymaticdomain.

In still another embodiment, a modified Clostridial toxin can comprisean amino to carboxyl single polypeptide linear order comprising anenzymatic domain, an exogenous protease cleavage site, a translocationdomain, and a non-Clostridial binding domain (FIG. 5A). In an aspect ofthis embodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxinenzymatic domain, an exogenous protease cleavage site, a Clostridialtoxin translocation domain, and a non-Clostridial binding domain.

In still another embodiment, a modified Clostridial toxin can comprisean amino to carboxyl single polypeptide linear order comprising atranslocation domain, an exogenous protease cleavage site, an enzymaticdomain and a non-Clostridial binding domain, (FIG. 5B). In an aspect ofthis embodiment, a modified Clostridial toxin can comprise an amino tocarboxyl single polypeptide linear order comprising a Clostridial toxintranslocation domain, a non-Clostridial binding domain, an exogenousprotease cleavage site and a Clostridial toxin enzymatic domain.

Aspects of the present specification provide, in part, an inactivationcleavage site. As used herein, the term “inactivation cleavage site”refers to a scissile bond together with adjacent or non-adjacentrecognition elements, or both, sufficient for selective proteolysis atthe scissile bond by a protease present in interstitial fluids orcirculatory systems, such as, e.g., the cardiovascular system or thelymphatic system. Such an inactivation cleavage site is operably-linkedas a fusion protein to a Clostridial toxin or Clostridial toxin chimericdisclosed in the present specification. By definition, an inactivationcleavage site is susceptible to selective cleavage by at least oneprotease present in interstitial fluids or circulatory systems.Non-limiting examples of inactivation cleavage sites include Thrombincleavage sites, Plasmin cleavage sites, Coagulation Factor VIIa cleavagesites, Coagulation Factor IXa cleavage sites, Coagulation Factor Xacleavage sites, Coagulation Factor XIa cleavage sites, CoagulationFactor XIIa cleavage sites, plasma kallikrein cleavage sites,protease-activated G protein-coupled receptor-1 (PAR1) cleavage sites,PAR 2 cleavage sites, PAR3 cleavage sites, PAR4 cleavage sites, MatrixMetalloproteinase-2 (MMP-2) cleavage sites, Matrix Metalloproteinase-9(MMP-9) cleavage sites, Furin cleavage sites, urokinase-type Plasminogenactivator (uPA) cleavage sites, tissue-type Plasminogen activator (tPA)cleavage sites, Tryptase-ε cleavage sites, Mouse mast cell protease-7(mMCP-7) cleavage sites, endothelin-converting enzyme-1 (ECE-1) cleavagesites, Kell blood group cleavage sites, DPPIV cleavage sites, ADAMmetallopeptidase with thrombospondin type 1 motif-13 (ADAMTS13) cleavagesites, and Cathepsin L cleavage sites (Table 4).

TABLE 4 Inactivation Cleavage Sites Reference SEQ Protease Cleavage SiteSequences ID NO: Thrombin LVPR*GS 114 LVPK*GS 115 FIPR*TF 116 VLPR*SF117 IVPR*SF 118 IVPR*GY 119 VVPR*GV 120 VLPR*LI 121 VMPR*SL 122 MFPR*SL123 Coagulation Factor VIIa (FVIIA) KLTR*AETV 125 DFTR*VVGG 126LSPR*TFHP 127 LIQR*NLSP 128 MATR*KMHD 129 LGIR*SFRN 130 PQGR*IVGG 131NLTR*IVGG 132 QVVR*IVGG 133 Coagulation Factor IXa (FIXa) PQGR*IVGG 135PQLR*MKNN 136 NLTR*IVGG 137 QVVR*IVGG 138 Coagulation Factor Xa (FXa)IDGR* 140 IEGR* 141 IDGR*SVGG 142 IDGR*TVGG 143 IDGR*IVGG 144 IEGR*SVGG145 IEGR*TVGG 146 IEGR*IVGG 147 PQGR*IVGG 148 IEGR*TSED 149 IEGR*IVEG150 IDGR*IVEG 151 FNPR*TFGS 152 FDER*TFGL 153 IDER*IVGG 154 FNEK*TFGL155 Coagulation Factor XIa (FXIa) AFWK*TDAS 157 KLTR*AETV 158 KLTR*AETI159 DFTR*VVGG 160 EFSR*VVGG 161 KLTR*AETV 162 DFTR*VVGG 163 IKPR*IVGG164 DLHR*HIFW 165 KQLR*VVNG 166 Coagulation Factor XIIa (FXIIa)PQGR*IVGG 168 IKPR*IVGG 169 SMTR*VVGG 170 TSTR*IVGG 171 PMKR*LTLG 172Kallikrein 1 SMTR*VVGG 174 SPFR*SSDI 175 SLMK*RPPG 176 YDWR*TPYL 177SPFR*SVQV 178 SPFR*TPYL 179 TFHK*AEYR 180 PRFK*IIGG 181 ISLM*KRPP 182LEAR*SAYH 183 EAKR*SYHS 184 PNRW*STGA 185 EAFY*SQFG 186 NAAR*STGA 187SSEW*SMPY 188 GTLF*RSGN 189 ARLY*SRGA 190 EASR*SATL 191 EASY*RRKQ 192TTFY*RRGA 193 AAWY*RTSR 194 SFHY*RMVG 195 ASSY*RTSR 196 TRFY*SRGR 197IKFF*SAQT 198 Protein C KKTR*NLKK 200 LDRR*GLQR 201 MATR*KMHD 202RLKK*SQFL 203 PQLR*MKNN 204 VDQR*GNQI 205 IEPR*SPSQ 206 KKTR*SPKT 207LDQR*GVQR 208 PDPR*SKNN 209 Plasminogen GEAR*GSVI 211 GHAR*LVHV 212AEFR*HDSG 213 HHQK*LVFF 214 GSNK*GALL 215 RAQR*SAGA 216 AFWK*TDAS 217MSMR*VRRH 218 RGVR*RTAS 219 RAAR*SQCT 220 PQSR*SVPP 221 PYLK*VFNP 222LSFR*ARAY 223 PQLR*RGWR 224 EDNR*DSSM 225 LSFR*ARAY 226 FRAR*AYGF 227YGFR*GPGP 228 ITFR*MNVA 229 THEK*GRQS 230 PRLK*ARAG 231 PKAK*SHAP 232PSHK*EGPQ 233 LFEK*KVYL 234 ADGK*KPSS 235 PRFK*IIGG 236 PQFR*IKGG 237PRCR*HRPH 238 KGYR*SQRG 239 DVAQ*FVLT 240 Matrix Metalloproteinase-2QPVS*VKVG 242 (MMP-2) RGVG*IKST 243 FVDC*LIEQ 244 VPAG*NWVL 245YHAD*IYDK 246 RACR*LAKA 247 QGAY*QEAF 248 DVLS*LLEK 249 TLDD*LIMA 250HISS*LIKL 251 DPNN*LLND 252 PVQP*QQSP 253 KPKT*ITGP 254 VVHP*LVLL 255HPLV*LLSV 256 AVAL*LIGP 257 QPLQ*LLDA 258 YIQG*INLV 259 LPQE*IKAN 260NISD*LTAA 261 KPRA*LTAL 262 APSW*LLTA 263 AVRW*LLTA 264 AVSW*LLTA 265SLRR*LTAA 266 SLSR*LTAL 267 RYSS*LTAA 268 SLAY*YTAL 269 SLRY*YTAA 270SPAY*YTAL 271 MHKA*LTAA 272 LRLA*ITAL 273 Matrix Metalloproteinase-9IPEN*FFGV 275 (MMP-9) MDIA*IHHP 276 SPSR*LFDQ 277 SEMR*LEKD 278FSVN*LDVK 279 RLFD*QFFG 280 FFGE*HLLE 281 GLSE*MRLE 282 SPEE*LKVK 283DVIE*VHGK 284 EVHG*KHEE 285 DEHG*FISR 286 GEHL*LESD 287 FHRK*YRIP 288GPRK*QVSG 289 LSPF*YLRP 290 PPSF*LRAP 291 NPLE*NSGF 292 VPYG*LGSP 293PPLK*LMHS 294 GPEG*LRVG 295 FMKG*LSKA 296 VVTG*VTAV 297 AIIG*LMVG 298SDLG*LTGI 299 VPYG*LGSP 300 GAAG*VKGD 301 GPTG*KQGD 302 GPSG*DQGA 303GPSG*FPFP 304 GAPG*FPGP 305 GAPG*NRGF 306 GLRG*ERGE 307 GPPG*SQGN 308GPAG*QQGA 309 GPPG*KDGT 310 GQPG*SPGS 311 GSPG*YQGP 312 GPVS*AVLT 313GPLG*MLSQ 314 GPLG*MWAQ 315 GPQG*IFGQ 316 LPRS*AKEL 317 NSFG*LRFG 318RAIH*INAE 319 Furin RPRR*AKRF 321 RKKR*GLYA 322 RERR*RKKR 323 RKKR*GLYA324 RKKR*TTSA 325 RHKR*ETLK 326 RLKR*DVVT 327 RMKR*EDLN 328 RAKR*FASL329 RKKR*FVSS 330 RTKR*FLSY 331 RRAR*SVDG 332 VFRR*DAHK 333 VFRR*EAHK334 RVAR*DITM 335 RISR*SLPQ 336 RSRR*AATS 337 RAKR*SPKH 338 FWHR*GVTK339 AKRR*TKRD 340 AKRR*AKRD 341 AKQR*AKRD 342 RDVR*GFAS 343 RKRR*SVNP344 RQKR*FVLS 345 RSKR*SLSC 346 u-Plasminogen Activator (u-PA) GSGK*SATL348 QRGR*SATL 349 RGSV*ILTV 350 PSSR*RRVN 351 CPGR*VVGG 352 PGAR*GRAF353 SSSR*GPTH 354 VSNK*YFSN 355 NSGR*AVTY 356 TYSR*SRYL 357 NSGR*AVTY358 PSGR*GRTL 359 AGSR*AVYY 360 TYGR*SRTN 361 NSSR*GVYL 362 PSSR*SVYN363 ASGR*GRTY 364 TSSR*AVYL 365 NSGR*SRTL 366 VSGR*IRTG 367 SSGR*IRTV368 t-Plasminogen Activator (t-PA) NALR*YAPD 370 CPGR*VVGG 371 PQFR*IKGG372 ALSR*MAVL 373 Tryptase-ε (Prosemin) *RVVGGE 375 *RIVGGE 376 *RIIGGE377 *RVVGGD 378 *RIVGGD 379 *RIIGGD 380 *KVVGGE 381 *KIVGGE 382 *KIIGGE383 *KVVGGD 384 *KIVGGD 385 *KIIGGD 386 Mouse mast cell protease-7LSSR*QSPG 388 (mMCP-7) LQAR*GASL 389 LGPK*AITM 390 LGPR*SAVY 391Endothelin-converting enzyme-1 HQKL*VFFA 393 (ECE-1) HHQK*LVFF 394KLVF*FAED 395 DRVY*IHPF 396 YIHP*FHLV 397 YGLG*SPRS 398 TPEH*VVPY 399DIIW*VNTP 400 DIIW*INTP 401 CHLD*IIWV 402 HLDI*IWVN 403 CVYF*CHLD 404SCSS*LMDK 405 ECVY*FCHL 406 RSKR*CSCS 407 RSKR*ALEN 408 GFSP*FRSS 409PRRP*YILP 410 KPQQ*FFGL 411 PQQF*FGLM 412Kell blood-group protein (KBGP) DIIW*VNTP 414 DIIW*INTP 415 Cathepsin LMFLE*AIPM 417 KVFQ*EPLF 418 ATLT*FDHS 419 PLFY*EAPR 420 TGLR*DPFN 421KILH*LPTS 422 AHLK*NSQE 423 APLT*AEIQ 424 EALF*AERK 425 EPLA*AERK 426GTFT*SDYS 427 KYLD*SRRA 428 QDFV*QWLM 429 KQLA*TKAA 430 STFE*ERSY 431LRLE*WPYQ 432 RGFF*YTPK 433 GFFY*TPKA 434 HFFK*NIVT 435 RGLS*LSRF 436QWLG*APVP 437 NMLK*RGLP 438 LSLA*HTHQ 439 TPFA*ATSS 440 KLLA*VSGP 441QLFR*RAVL 442 PRFK*IIGG 443 PAR1 *SFLLRN 445 *SFFLRN 446 *SFFLKN 447*TFLLRN 448 *GFPGKF 449 *GYPAKF 450 *GYPLKF 451 *GYPIKF 452 PAR2 *SLIGKV454 *SLIGRL 455 PAR3 *TFRGAP 457 *SFNGGP 458 *SFNGNE 459 PAR4 *GYPGQV461 *AYPGKF 462 *TYPGKF 463 *GYPGKY 464 *GYPGKW 465 *GYPGKK 466 *GYPLKF467 *GYPGRF 468 *GYPGFK 469 *GYPAKF 470 *GFPGKF 471 *GFPGKP 472 *SYPGKF473 *SYPAKF 474 *SYPGRF 475 *SYAGKF 476 *SFPGQP 477 *SFPGQA 478 ADAMTS13NLVY*MVTG 479 An asterisks (*) indicates the peptide bond of the P₁-P₁cleavage site that is cleaved by the indicated protease.

It is envisioned that an inactivation cleavage site of any and alllengths can be useful in aspects of the present specification with theproviso that the inactivation cleavage site is capable of being cleavedby a interstitial fluid or circulatory system protease. Thus, in aspectsof this embodiment, an inactivation cleavage site can be, e.g., at least3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acids in length. In otheraspects of this embodiment, an inactivation cleavage site can be, e.g.,at most 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acids in length.

An inactivation cleavage site useful in aspects of the specificationincludes, without limitation, naturally occurring inactivation cleavagesite; naturally occurring inactivation cleavage site variants; andnon-naturally-occurring inactivation cleavage site variants, such as,e.g., conservative inactivation cleavage site variants, non-conservativeinactivation cleavage site variants and inactivation cleavage sitepeptidomimetics. As used herein, the term “inactivation cleavage sitevariant,” whether naturally-occurring or non-naturally-occurring, refersto an inactivation cleavage site that has at least one amino acid changefrom the corresponding region of the disclosed reference sequences andcan be described in percent identity to the corresponding region of thatreference sequence. Any of a variety of sequence alignment methods canbe used to determine percent identity, including, without limitation,global methods, local methods and hybrid methods, such as, e.g., segmentapproach methods. Protocols to determine percent identity are routineprocedures within the scope of one skilled in the art and from theteaching herein.

As used herein, the term “naturally occurring inactivation cleavage sitevariant” refers to any inactivation cleavage site produced without theaid of any human manipulation. Non-limiting examples of inactivationcleavage sites include Thrombin cleavage site variants, Plasmin cleavagesite variants, Coagulation Factor V cleavage site variants, CoagulationFactor VII cleavage site variants, Coagulation Factor VIII cleavage sitevariants, Coagulation Factor IXa cleavage site variants, CoagulationFactor Xa cleavage site variants, Coagulation Factor XIa cleavage sitevariants, Coagulation Factor XIIa cleavage site variants, plasmakallikrein cleavage site variants, MMP-2 cleavage site variants, MMP-9cleavage site variants, Furin cleavage site variants, u-Plasminogenactivator cleavage site variants, t-Plasminogen activator cleavage sitevariants, Tryptase-ε cleavage site variants, mMCP-7 cleavage sitevariants, ECE-1 cleavage site variants, KBGP cleavage site variants,Cathepsin L cleavage site variants, PAR1 cleavage site variants, PAR2cleavage site variants, PAR3 cleavage site variants, PAR4 cleavage sitevariants, and ADAM-TS13 cleavage site variants.

As used herein, the term “non-naturally occurring inactivation cleavagesite variant” refers to any inactivation cleavage site produced with theaid of human manipulation, including, without limitation, inactivationcleavage site variants produced by genetic engineering using randommutagenesis or rational design and inactivation cleavage site variantsproduced by chemical synthesis. Non-limiting examples of non-naturallyoccurring inactivation cleavage site variants include, e.g.,conservative inactivation cleavage site variants, non-conservativeinactivation cleavage site variants, and inactivation cleavage sitepeptidomimetics.

As used herein, the term “conservative inactivation cleavage sitevariant” refers to an inactivation cleavage site that has at least oneamino acid substituted by another amino acid or an amino acid analogthat has at least one property similar to that of the original aminoacid from the reference inactivation cleavage site sequence. Examples ofproperties include, without limitation, similar size, topography,charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bondingcapacity, hydrogen-bonding capacity, a physicochemical property, of thelike, or any combination thereof. A conservative inactivation cleavagesite variant can function in substantially the same manner as thereference inactivation cleavage site on which the conservativeinactivation cleavage site variant is based, and can be substituted forthe reference inactivation cleavage site in any aspect of the presentspecification. Non-limiting examples of a conservative inactivationcleavage site variant include, e.g., conservative Thrombin cleavage sitevariants, conservative Plasmin cleavage site variants, conservativeCoagulation Factor V cleavage site variants, conservative CoagulationFactor VII cleavage site variants, conservative Coagulation Factor VIIIcleavage site variants, conservative Coagulation Factor IXa cleavagesite variants, conservative Coagulation Factor Xa cleavage sitevariants, conservative Coagulation Factor XIa cleavage site variants,conservative Coagulation Factor XIIa cleavage site variants,conservative plasma kallikrein cleavage site variants, conservativeMMP-2 cleavage site variants, conservative MMP-9 cleavage site variants,conservative Furin cleavage site variants, conservative u-Plasminogenactivator cleavage site variants, conservative t-Plasminogen activatorcleavage site variants, conservative Tryptase-ε cleavage site variants,conservative mMCP-7 cleavage site variants, conservative ECE-1 cleavagesite variants, conservative KBGP cleavage site variants, conservativeCathepsin L cleavage site variants, conservative PAR1 cleavage sitevariants, conservative PAR2 cleavage site variants, conservative PAR3cleavage site variants, conservative PAR4 cleavage site variants, andconservative ADAM-TS13 cleavage site variants.

As used herein, the term “non-conservative inactivation cleavage sitevariant” refers to an inactivation cleavage site in which 1) at leastone amino acid is deleted from the reference inactivation cleavage siteon which the non-conservative inactivation cleavage site variant isbased; 2) at least one amino acid added to the reference inactivationcleavage site on which the non-conservative inactivation cleavage siteis based; or 3) at least one amino acid is substituted by another aminoacid or an amino acid analog that does not share any property similar tothat of the original amino acid from the reference inactivation cleavagesite sequence (Table 4). A non-conservative inactivation cleavage sitevariant can function in substantially the same manner as the referenceinactivation cleavage site on which the non-conservative inactivationcleavage site is based, and can be substituted for the referenceinactivation cleavage site in any aspect of the present specification.Non-limiting examples of a non-conservative inactivation cleavage sitevariant include, e.g., non-conservative Thrombin cleavage site variants,non-conservative Plasmin cleavage site variants, non-conservativeCoagulation Factor V cleavage site variants, non-conservativeCoagulation Factor VII cleavage site variants, non-conservativeCoagulation Factor VIII cleavage site variants, non-conservativeCoagulation Factor IXa cleavage site variants, non-conservativeCoagulation Factor Xa cleavage site variants, non-conservativeCoagulation Factor XIa cleavage site variants, non-conservativeCoagulation Factor XIIa cleavage site variants, non-conservative plasmakallikrein cleavage site variants, non-conservative MMP-2 cleavage sitevariants, non-conservative MMP-9 cleavage site variants,non-conservative Furin cleavage site variants, non-conservativeu-Plasminogen activator cleavage site variants, non-conservativet-Plasminogen activator cleavage site variants, non-conservativeTryptase-ε cleavage site variants, non-conservative mMCP-7 cleavage sitevariants, non-conservative ECE-1 cleavage site variants,non-conservative KBGP cleavage site variants, non-conservative CathepsinL cleavage site variants, non-conservative PAR1 cleavage site variants,non-conservative PAR2 cleavage site variants, non-conservative PAR3cleavage site variants, non-conservative PAR4 cleavage site variants,and non-conservative ADAM-TS13 cleavage site variants.

As used herein, the term “inactivation cleavage site peptidomimetic”refers to an inactivation cleavage site that has at least one amino acidsubstituted by a non-natural oligomer that has at least one propertysimilar to that of the first amino acid. Examples of properties include,without limitation, topography of a peptide primary structural element,functionality of a peptide primary structural element, topology of apeptide secondary structural element, functionality of a peptidesecondary structural element, of the like, or any combination thereof.An inactivation cleavage site peptidomimetic can function insubstantially the same manner as the reference inactivation cleavagesite on which the inactivation cleavage site peptidomimetic is based,and can be substituted for the reference inactivation cleavage site inany aspect of the present specification. For examples of peptidomimeticmethods see, e.g., Amy S. Ripka & Daniel H. Rich, Peptidomimetic design,2(4) CURR. OPIN. CHEM. BIOL. 441-452 (1998); and M. Angels Estiarte &Daniel H. Rich, Peptidomimetics for Drug Design, 803-861 (BURGER'SMEDICINAL CHEMISTRY AND DRUG DISCOVERY VOl. 1 PRINCIPLE AND PRACTICE,Donald J. Abraham ed., Wiley-Interscience, 6^(th) ed 2003). Non-limitingexamples of an inactivation cleavage site peptidomimetic include, e.g.,Thrombin cleavage site peptidomimetics, Plasmin cleavage sitepeptidomimetics, Coagulation Factor V cleavage site peptidomimetics,Coagulation Factor VII cleavage site peptidomimetics, Coagulation FactorVIII cleavage site peptidomimetics, Coagulation Factor IXa cleavage sitepeptidomimetics, Coagulation Factor Xa cleavage site peptidomimetics,Coagulation Factor XIa cleavage site peptidomimetics, Coagulation FactorXIIa cleavage site peptidomimetics, plasma kallikrein cleavage sitepeptidomimetics, MMP-2 cleavage site peptidomimetics, MMP-9 cleavagesite peptidomimetics, Furin cleavage site peptidomimetics, u-Plasminogenactivator cleavage site peptidomimetics, t-Plasminogen activatorcleavage site peptidomimetics, Tryptase-ε cleavage site peptidomimetics,mMCP-7 cleavage site variants, ECE-1 cleavage site peptidomimetics, KBGPcleavage site peptidomimetics, Cathepsin L cleavage sitepeptidomimetics, PAR1 cleavage site peptidomimetics, PAR2 cleavage sitepeptidomimetics, PAR3 cleavage site peptidomimetics, PAR4 cleavage sitepeptidomimetics, and ADAM-TS13 cleavage site peptidomimetics.

Thus, in an embodiment, a Clostridial toxin comprises an inactivationcleavage site. In an aspect of this embodiment, a Clostridial toxincomprises a Clostridial toxin enzymatic domain, a Clostridial toxintranslocation domain, a Clostridial toxin binding domain, and aninactivation cleavage site. In another aspect of this embodiment, aClostridial toxin comprises a naturally occurring inactivation cleavagesite variant, such as, e.g., an inactivation cleavage site isoform. Inanother aspect of this embodiment, a Clostridial toxin comprises anon-naturally occurring inactivation cleavage site variant, such as,e.g., a conservative inactivation cleavage site variant, anon-conservative inactivation cleavage site variant or an activeinactivation cleavage site fragment, or any combination thereof.

In another embodiment, a Clostridial toxin chimeric comprises aninactivation cleavage site. In an aspect of this embodiment, aClostridial toxin chimeric comprises a Clostridial toxin enzymaticdomain, a Clostridial toxin translocation domain, a non-Clostridialtoxin binding domain, and an inactivation cleavage site. In anotheraspect of this embodiment, a Clostridial toxin chimeric comprises anaturally occurring inactivation cleavage site variant, such as, e.g.,an inactivation cleavage site isoform. In another aspect of thisembodiment, a Clostridial toxin chimeric comprises a non-naturallyoccurring inactivation cleavage site variant, such as, e.g., aconservative inactivation cleavage site variant, a non-conservativeinactivation cleavage site variant or an active inactivation cleavagesite fragment, or any combination thereof.

In another embodiment, a hydrophobic amino acid at one particularposition in the inactivation cleavage site can be substituted withanother hydrophobic amino acid. Examples of hydrophobic amino acidsinclude, e.g., C, F, I, L, M, V and W. In another aspect of thisembodiment, an aliphatic amino acid at one particular position in theinactivation cleavage site can be substituted with another aliphaticamino acid. Examples of aliphatic amino acids include, e.g., A, I, L, P,and V. In yet another aspect of this embodiment, an aromatic amino acidat one particular position in the inactivation cleavage site can besubstituted with another aromatic amino acid. Examples of aromatic aminoacids include, e.g., F, H, W and Y. In still another aspect of thisembodiment, a stacking amino acid at one particular position in theinactivation cleavage site can be substituted with another stackingamino acid. Examples of stacking amino acids include, e.g., F, H, W andY. In a further aspect of this embodiment, a polar amino acid at oneparticular position in the inactivation cleavage site can be substitutedwith another polar amino acid. Examples of polar amino acids include,e.g., D, E, K, N, Q, and R. In a further aspect of this embodiment, aless polar or indifferent amino acid at one particular position in theinactivation cleavage site can be substituted with another less polar orindifferent amino acid. Examples of less polar or indifferent aminoacids include, e.g., A, H, G, P, S, T, and Y. In a yet further aspect ofthis embodiment, a positive charged amino acid at one particularposition in the inactivation cleavage site can be substituted withanother positive charged amino acid. Examples of positive charged aminoacids include, e.g., K, R, and H. In a still further aspect of thisembodiment, a negative charged amino acid at one particular position inthe inactivation cleavage site can be substituted with another negativecharged amino acid. Examples of negative charged amino acids include,e.g., D and E. In another aspect of this embodiment, a small amino acidat one particular position in the inactivation cleavage site can besubstituted with another small amino acid. Examples of small amino acidsinclude, e.g., A, D, G, N, P, S, and T. In yet another aspect of thisembodiment, a C-beta branching amino acid at one particular position inthe inactivation cleavage site can be substituted with another C-betabranching amino acid. Examples of C-beta branching amino acids include,e.g., I, T and V.

Aspects of the present specification disclose, in part, a Thrombincleavage site as an inactivation cleavage site. As used herein, the term“Thrombin cleavage site” refers to a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by Thrombin under conditionssuitable for Thrombin protease activity. It is envisioned that any aminoacid sequence cleaved by Thrombin can be useful in aspects of thepresent specification. Although exceptions are known, a generalizedconsensus sequence for a Thrombin cleavage site is X₁X₂X₃(R/K)*X₄X₅X₆X₇(SEQ ID NO: 113), where X₁ is preferentially S, T, an amidic amino acidlike N and Q, a positive amino acid like H, K, and R, an aromatichydrophobic amino acid like F, W, and Y, an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₂ is any amino acid; X₃ ispreferentially F, S, T, an amidic amino acid like N or Q, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ ispreferentially S, T, a positive amino acid like H, K, and R, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; and X₅,X₆, and X₇, are independently any amino acid. Table 4 lists exemplaryreference cleavage sites for Thrombin (SEQ ID NO: 114-123). AdditionalThrombin cleavage sites are well known in the art or can be defined byroutine methods. See, e.g., O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Thrombin cleavage site. In an aspect of thisembodiment, a Thrombin cleavage site comprises the consensus sequenceSEQ ID NO: 113, where X₁ is S, T, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, an aromatic hydrophobic amino acidlike F, W, and Y, an aliphatic hydrophobic amino acid like, G, P, A, V,L, I, and M; X₂ is any amino acid; X₃ is F, S, T, an amidic amino acidlike N or Q, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₄ is S, T, a positive amino acid like H, K, and R, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; and X₅,X₆, and X₇, are independently any amino acid. In another aspect of thisembodiment, a Thrombin cleavage site comprises the consensus sequenceSEQ ID NO: 113, where X₁ is S, Q, K, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₂ is an acidic amino acid like Dand E, an amidic amino acid like N and Q, a basic amino acid like K andR, an uncharged amino acid like C, S, and T, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₃ is N, Q, G, P, A, V, L, orI; X₄ is S, T, H, G, A, L, or I; X₅ is S, T, Q, K, R, F, Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ is S,T, Q, K, R, G, P, A, V, L, or I; and X₇ is S, T, Q, K, R, G, P, A, V, L,or I. In another aspect of this embodiment, a Thrombin cleavage sitecomprises the consensus sequence SEQ ID NO: 113, where X₁ is Q, G, P, A,V, L, I, or M; X₂ is S, T, D, E, G, A, V, or I; X₃ is G, P, A, V, or L;X₄ is S, G, A, or L; X₅ is Q, K, F, A, V, or L; X₆ is S, Q, K, R, G, P,V, or L; and X₇ is S, T, K, G, V, L, or I. In other aspects of thisembodiment, a Thrombin cleavage site comprises, e.g., SEQ ID NO: 114,SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ IDNO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, or SEQ ID NO:123.

Aspects of the present specification disclose, in part, a Plasmincleavage site as an inactivation cleavage site. As used herein, the term“Plasmin cleavage site” refers to a scissile bond together with adjacentor non-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by Plasmin under conditions suitablefor Plasmin protease activity. It is envisioned that any amino acidsequence cleaved by Plasmin can be useful in aspects of the presentspecification. Plasmin catalyzes cleavage of Lys| and Arg| bonds, with aspecificity similar to that of Trypsin. However, Plasmin is a much lessefficient enzyme than Trypsin, and cleaves only some of these bonds inproteins. Trypsin cleaves peptide chains mainly at the carboxyl side ofthe amino acids lysine or arginine, except when either is followed byproline.

Aspects of the present specification disclose, in part, a CoagulationFactor VIIa cleavage site as an inactivation cleavage site. As usedherein, the term “Coagulation Factor VIIa cleavage site” or “FVIIacleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by FVIIa under conditions suitable forFVIIa protease activity. It is envisioned that any amino acid sequencecleaved by FVIIa can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aFVIIa cleavage site is X₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 124), where X₁is preferentially an acidic amino acid like D and E, an amidic aminoacid like N and Q, a basic amino acid like K and R, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₂ is Q, S, T, anaromatic hydrophobic amino acid like F, W and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ ispreferentially Q, S, T, or an aliphatic hydrophobic amino acid like, G,P, A, V, L, I, and M; X₄, X₅, X₆, and X₇, are independently any aminoacid. Table 4 lists exemplary reference cleavage sites for FVIIa (SEQ IDNO: 125-133). Additional FVIIa cleavage sites are well known in the artor can be defined by routine methods. See, e.g., J. H. Morrissey,Coagulation Factor VIIa. In HANDBOOK OF PROTEOLYTIC ENZYMES, pp.1659-1662 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds;Elsevier, London, 2d, 2004); O, Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Coagulation Factor VIIa cleavage site. In an aspectof this embodiment, a Coagulation Factor VIIa cleavage site comprisesthe consensus sequence SEQ ID NO: 124, where X₁ is an acidic amino acidlike D and E, an amidic amino acid like N and Q, a basic amino acid likeK and R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is Q, S, T, an aromatic hydrophobic amino acid like F, W andY, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;X₃ is Q, S, T, or an aliphatic hydrophobic amino acid like, G, P, A, V,L, I, and M; and X₄, X₅, X₆, and X₇, are independently any amino acid.In other aspects of this embodiment, a Coagulation Factor VIIa cleavagesite comprises, e.g., SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127,SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ IDNO: 132, or SEQ ID NO: 133

Aspects of the present specification disclose, in part, a CoagulationFactor IXa cleavage site as an inactivation cleavage site. As usedherein, the term “Coagulation Factor IXa cleavage site” or “FIXacleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by FIXa under conditions suitable forFIXa protease activity. It is envisioned that any amino acid sequencecleaved by FIXa can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aFIXa cleavage site is X₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 134), where X₁ ispreferentially an acidic amino acid like D and E, an amidic amino acidlike N and Q, or an aliphatic hydrophobic amino acid like, G, P, A, V,L, I, and M; X₂ is preferentially an acidic amino acid like D and E, anamidic amino acid like N and Q, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₃ is preferentially, S, T, an aromatichydrophobic amino acid like F, W and Y, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; and X₄, X₅, X₆, and X₇, areindependently any amino acid. Table 4 lists exemplary reference cleavagesites for FIXa (SEQ ID NO: 135-138). Additional FIXa cleavage sites arewell known in the art or can be defined by routine methods. See, e.g.,A. T. Thompson, Molecular Biology of Factor IX. In HEMOSTASIS ANDTHROMBOSIS, BASIC PRINCIPLES AND CLINICAL PRACTICE, pp. 128-129 (R. W.Colman, J. Hirsh, V. J. Marder, A. W Clowes, J. N. George, eds;Lippincott Williams & Wilkins, Philadelphia, Pa., 2d, 2001); S. Kawabataand S. Iwanaga, Russellysin. In HANDBOOK OF PROTEOLYTIC ENZYMES, pp.683-684 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds;Elsevier, London, 2d, 2004); A. E. Schmidt and S. P. Bajaj, Coagulationfactor IXa. In HANDBOOK OF PROTEOLYTIC ENZYMES, pp. 1655-1659 (A. J.Barrett, N. D. Rawlings, and J. F. Woessner, eds; Elsevier, London, 2d,2004); O. Schilling and C. M. Overall, Proteome-Derived,Database-Searchable Peptide Libraries for Identifying Protease CleavageSites, Nat. Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al.,MEROPS: The Peptidase Database, Nucleic Acids Res. 36(Database issue):D320-D325 (2008); Neil D. Rawlings, et al., MEROPS: The PeptidaseDatabase, Nucleic Acids Res. 38(Database issue): D227-D233 (2010); NeilD. Rawlings, et al., A Large and Accurate Collection of PeptidaseCleavages in the MEROPS Database, Database in press (2010), each ofwhich is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Coagulation Factor IXa cleavage site. In an aspectof this embodiment, a Coagulation Factor IXa cleavage site comprises theconsensus sequence SEQ ID NO: 134, where X₁ is an acidic amino acid likeD and E, an amidic amino acid like N and Q, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₂ is an acidic amino acidlike D and E, an amidic amino acid like N and Q, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is, S, T, anaromatic hydrophobic amino acid like F, W and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₄, X₅, X₆,and X₇, are independently any amino acid. In another aspect of thisembodiment, a Coagulation Factor IXa cleavage site comprises theconsensus sequence SEQ ID NO: 134, where X₁ is an acidic amino acid likeD and E, an amidic amino acid like N and Q, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₂ is an acidic amino acidlike D and E, an amidic amino acid like N and Q, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is, S, T, anaromatic hydrophobic amino acid like F, W and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₄, X₅, X₆, andX₇, are independently an acidic amino acid like D and E, an amidic aminoacid like N and Q, or an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M. In other aspects of this embodiment, a CoagulationFactor IXa cleavage site comprises, e.g., SEQ ID NO: 135, SEQ ID NO:136, SEQ ID NO: 137, or SEQ ID NO: 138.

Aspects of the present specification disclose, in part, a CoagulationFactor Xa cleavage site as an inactivation cleavage site. As usedherein, the term “Coagulation Factor Xa cleavage site” or “FXa cleavagesite” refers to a scissile bond together with adjacent or non-adjacentrecognition elements, or both, sufficient for detectable proteolysis atthe scissile bond by FXa under conditions suitable for FXa proteaseactivity. It is envisioned that any amino acid sequence cleaved by FXacan be useful in aspects of the present specification. Althoughexceptions are known, a generalized consensus sequence for a FXacleavage site is X₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 139), where X₁ is anyamino acid, X₂ is preferentially G, A, S, an acidic amino acid like Dand E, an amidic amino acid like Q and N, or an aromatic hydrophobicamino acid like F, W and Y, X₃ is preferentially an aromatic hydrophobicamino acid like F, W and Y, or an aliphatic hydrophobic amino acid like,G, P, A, V, L, I, and M; X₄ is preferentially an amidic amino acid likeN and Q, an uncharged amino acid like C, S, and T, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₅, X₆, and X₇,are independently any amino acid. Table 4 lists exemplary referencecleavage sites for FXa (SEQ ID NO: 140-155). Additional FXa cleavagesites are well known in the art or can be defined by routine methods.See, e.g., D. L. Greenberg and E. W. Davie, Blood Coagulation Factors:Their Complementary DNAs, Genes, and Expression. In HEMOSTASIS ANDTHROMBOSIS, BASIC PRINCIPLES AND CLINICAL PRACTICE, pp. 34-35 (R. W.Colman, J. Hirsh, V. J. Marder, A. W Clowes, J. N. George, eds;Lippincott Williams & Wilkins, Philadelphia, Pa., 2d, 2001); O.Schilling and C. M. Overall, Proteome-Derived, Database-SearchablePeptide Libraries for Identifying Protease Cleavage Sites, Nat.Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: ThePeptidase Database, Nucleic Acids Res. 36(Database issue): D320-D325(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,Nucleic Acids Res. 38(Database issue): D227-D233 (2010); Neil D.Rawlings, et al., A Large and Accurate Collection of Peptidase Cleavagesin the MEROPS Database, Database in press (2010), each of which isincorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Coagulation Factor Xa cleavage site. In an aspectof this embodiment, a Coagulation Factor Xa cleavage site comprises theconsensus sequence SEQ ID NO: 139, where X₁ is any amino acid, X₂ is G,A, S, an acidic amino acid like D and E, an amidic amino acid like Q andN, or an aromatic hydrophobic amino acid like F, W and Y, X₃ is anaromatic hydrophobic amino acid like F, W and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ is an amidicamino acid like N and Q, an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₅, X₆,and X₇, are independently any amino acid. In another aspect of thisembodiment, a Coagulation Factor Xa cleavage site comprises theconsensus sequence SEQ ID NO: 139, where X₁ is E, F, P, A, L, or I; X₂is S, Q, D, E, F, G, or A; X₃ is F, G, or P; X₄ is S, T, L, or I; X₅ isS, F, A, or V; X₆ is S, T, E, N, H, G, A, or M; and X₇ is S, N, D, Q, K,R, or G. In another aspect of this embodiment, a Coagulation Factor Xacleavage site comprises the consensus sequence SEQ ID NO: 139, where X₁is I or A; X₂ is E or F; X₃ is F, G, or P; is S, T, or I; X₅ is S, F, orV; X₆ is E or G; and X₇ is S or G. In other aspects of this embodiment,a Coagulation Factor Xa cleavage site comprises, e.g., the amino acidsequence SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144,SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ IDNO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153,SEQ ID NO: 154, or SEQ ID NO: 155.

Aspects of the present specification disclose, in part, a CoagulationFactor XIa cleavage site as an inactivation cleavage site. As usedherein, the term “Coagulation Factor XIa cleavage site” or “FXIacleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by FXIa under conditions suitable forFXIa protease activity. It is envisioned that any amino acid sequencecleaved by FXIa can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aFXIa cleavage site is X₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 156), where X₁ ispreferentially an acidic amino acid like D or E, a basic amino acid likeK and R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is preferentially an acidic amino acid like D or E, an amidicamino acid like Q and N, a basic amino acid like K and R, an aromatichydrophobic amino acid like F, W and Y, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₃ is preferentially H, anuncharged amino acid like C, S, and T, an aromatic hydrophobic aminoacid like F, W and Y, or an aliphatic hydrophobic amino acid like, G, P,A, V, L, I, and M; X₄ is preferentially H, an uncharged amino acid likeC, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₅ is preferentially an acidic amino acid like D and E, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ ispreferentially an amidic amino acid like Q and N, an uncharged aminoacid like C, S, and T, an aromatic hydrophobic amino acid like F, W andY, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;and X₇ is preferentially an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M. Table 4lists exemplary reference cleavage sites for FXIa (SEQ ID NO: 157-166).Additional FXIa cleavage sites are well known in the art or can bedefined by routine methods. See, e.g., P. N. Walsh, Coagulation FactorXIa. In Handbook of Proteolytic Enzymes, pp. 1651-1655 (A. J. Barrett,N. D. Rawlings, and J. F. Woessner, eds; Elsevier, London, 2d, 2004); O.Schilling and C. M. Overall, Proteome-Derived, Database-SearchablePeptide Libraries for Identifying Protease Cleavage Sites, Nat.Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: ThePeptidase Database, Nucleic Acids Res. 36(Database issue): D320-D325(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,Nucleic Acids Res. 38(Database issue): D227-D233 (2010); Neil D.Rawlings, et al., A Large and Accurate Collection of Peptidase Cleavagesin the MEROPS Database, Database in press (2010), each of which isincorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Coagulation Factor XIa cleavage site. In an aspectof this embodiment, a Coagulation Factor XIa cleavage site comprises theconsensus sequence SEQ ID NO: 156, where X₁ is an acidic amino acid likeD or E, a basic amino acid like K and R, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₂ is an acidic amino acidlike D or E, an amidic amino acid like Q and N, a basic amino acid likeK and R, an aromatic hydrophobic amino acid like F, W and Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is H,an uncharged amino acid like C, S, and T, an aromatic hydrophobic aminoacid like F, W and Y, or an aliphatic hydrophobic amino acid like, G, P,A, V, L, I, and M; X₄ is H, an uncharged amino acid like C, S, and T, oran aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₅ isan acidic amino acid like D and E, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₆ is an amidic amino acid like Qand N, an uncharged amino acid like C, S, and T, an aromatic hydrophobicamino acid like F, W and Y, or an aliphatic hydrophobic amino acid like,G, P, A, V, L, I, and M; and X₇ is an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M. In another aspect of this embodiment, a Coagulation Factor XIacleavage site comprises the consensus sequence SEQ ID NO: 156, where X₁is an acidic amino acid like D or E, or a basic amino acid like K and R;X₂ is an aromatic hydrophobic amino acid like F, W and Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is anuncharged amino acid like C, S, and T, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₄ is an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₅ is an acidic amino acid like Dand E, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₆ is an uncharged amino acid like C, S, and T, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₇ is analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M. Inanother aspect of this embodiment, a Coagulation Factor XIa cleavagesite comprises the consensus sequence SEQ ID NO: 156, where X₁ is D orK; X₂ is F or L; X₃ is T or P; X₄ is A or V; X₅ is E or V; X₆ is T or G;and X₇ is G or V. In other aspects of this embodiment, a CoagulationFactor XIa cleavage site comprises, e.g., SEQ ID NO: 157, SEQ ID NO:158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, or SEQ ID NO: 166.

Aspects of the present specification disclose, in part, a CoagulationFactor XIIa cleavage site as an inactivation cleavage site. As usedherein, the term “Coagulation Factor XIIa cleavage site” or “FXIIacleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by FXIIa under conditions suitable forFXIIa protease activity. It is envisioned that any amino acid sequencecleaved by FXIIa can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aFXIIa cleavage site is X₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 167), where X₁is preferentially an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₂ ispreferentially an acidic amino acid like D and E, a basic amino acidlike K and R, an uncharged amino acid like C, S, and T, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ ispreferentially a basic amino acid like K and R, an uncharged amino acidlike C, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M; X₄ is preferentially an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₅ is preferentially an uncharged aminoacid like C, S, and T, or an aliphatic hydrophobic amino acid like, G,P, A, V, L, I, and M; X₆ is preferentially an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; and X₇ is preferentially analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M. Table 4lists exemplary reference cleavage sites for FXIIa (SEQ ID NO: 168-172).Additional FXIIa cleavage sites are well known in the art or can bedefined by routine methods. See, e.g., O. D. Ratnoff, Coagulation FactorXIIa. In Handbook of Proteolytic Enzymes, pp. 1642-1644 (A. J. Barrett,N. D. Rawlings, and J. F. Woessner, eds; Elsevier, London, 2d, 2004); O.Schilling and C. M. Overall, Proteome-Derived, Database-SearchablePeptide Libraries for Identifying Protease Cleavage Sites, Nat.Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: ThePeptidase Database, Nucleic Acids Res. 36(Database issue): D320-D325(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,Nucleic Acids Res. 38(Database issue): D227-D233 (2010); Neil D.Rawlings, et al., A Large and Accurate Collection of Peptidase Cleavagesin the MEROPS Database, Database in press (2010), each of which isincorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Coagulation Factor XIIa cleavage site. In an aspectof this embodiment, a Coagulation Factor XIIa cleavage site comprisesthe consensus sequence SEQ ID NO: 167, where X₁ is an uncharged aminoacid like C, S, and T, or an aliphatic hydrophobic amino acid like, G,P, A, V, L, I, and M; X₂ is an acidic amino acid like D and E, a basicamino acid like K and R, an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is abasic amino acid like K and R, an uncharged amino acid like C, S, and T,or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄is an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₅is an uncharged amino acid like C, S, and T, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₆ is an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; and X₇ is an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M. In other aspect ofthis embodiment, a Coagulation Factor XIIa cleavage site comprises theconsensus sequence SEQ ID NO: 167, where X₁ is S, T, P, or I; X₂ is Q,K, S, or M; X₃ is K, T, G, or P; X₄ is L, I, or V; X₅ is T or V; X₆ is Gor L; and X₇ is G. In other aspects of this embodiment, a CoagulationFactor XIIa cleavage site comprises, e.g., SEQ ID NO: 168, SEQ ID NO:169, SEQ ID NO: 170, SEQ ID NO: 171, or SEQ ID NO: 172.

Aspects of the present specification disclose, in part, a Kallikrein 1cleavage site as an inactivation cleavage site. As used herein, the term“Kallikrein 1 cleavage site” refers to a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by Kallikrein 1 underconditions suitable for Kallikrein 1 protease activity. It is envisionedthat any amino acid sequence cleaved by Kallikrein 1 can be useful inaspects of the present specification. Although exceptions are known, ageneralized consensus sequence for a Kallikrein 1 cleavage site isX₁X₂X₃X₄*(R/K/S)X₅X₆X₇ (SEQ ID NO: 173), where X₁ is preferentially anacidic amino acid like D and E, an amidic amino acid like Q and N, anuncharged amino acid like C, S, and T, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₂ is any amino acid; X₃ is anyamino acid; X₄ is preferentially a positive amino acid like H, K, and R,a large non-polar amino acid like F, I, L, M and V, or an aromatichydrophobic amino acid like F, W and Y; X₅ is any amino acid; X₆ is anyamino acid; and X₇ is any amino acid. Table 4 lists exemplary referencecleavage sites for Kallikrein 1 (SEQ ID NO: 174-198). AdditionalKallikrein 1 cleavage sites are well known in the art or can be definedby routine methods. See, e.g., R. W. Colman, Contact Activation Pathway:Inflammation, Fibrinolytic, Anticoagulant, Antiadhesive, andAntiangiogenic Activities. In HEMOSTASIS AND THROMBOSIS, BASICPRINCIPLES AND CLINICAL PRACTICE, pp. 103-104 (R. W. Colman, J. Hirsh,V. J. Marder, A. W Clowes, J. N. George, eds; Lippincott Williams &Wilkins, Philadelphia, Pa., 2d, 2001); J. Chao, Human Kallikrein 1,Tissue Kallikrein. In Handbook of Proteolytic Enzymes, pp. 1577-1580 (A.J. Barrett, N. D. Rawlings, and J. F. Woessner, eds; Elsevier, London,2d, 2004); H. X. Li, et al., Substrate Specificity of Human Kallikreins1 and 6 Determined by Phage Display, Protein Sci. 17: 664-672 (2008); O.Schilling and C. M. Overall, Proteome-Derived, Database-SearchablePeptide Libraries for Identifying Protease Cleavage Sites, Nat.Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: ThePeptidase Database, Nucleic Acids Res. 36(Database issue): D320-D325(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,Nucleic Acids Res. 38(Database issue): D227-D233 (2010); Neil D.Rawlings, et al., A Large and Accurate Collection of Peptidase Cleavagesin the MEROPS Database, Database in press (2010), each of which isincorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Kallikrein 1 cleavage site. In an aspect of thisembodiment, a Kallikrein 1 cleavage site comprises the consensussequence SEQ ID NO: 173, where X₁ is an acidic amino acid like D and E,an amidic amino acid like Q and N, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is any amino acid; X₃ is any amino acid; X₄ is a positiveamino acid like H, K, and R, a large non-polar amino acid like F, I, L,M and V, or an aromatic hydrophobic amino acid like F, W and Y; X₅ isany amino acid; X₆ is any amino acid; and X₇ is any amino acid. Inanother aspect of this embodiment, a Kallikrein 1 cleavage sitecomprises the consensus sequence SEQ ID NO: 173, where X₁ is D, S, T, oran aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₂ isS, T, A, P, or V; X₃ is S, F, or L; X₄ is R or an aromatic hydrophobicamino acid like F, W and Y; X₅ is R, S, T, or A; X₆ is R, S, or G; andX₇ is R, G, or A. In other aspects of this embodiment, a Kallikrein 1cleavage site comprises, e.g., SEQ ID NO: 174, SEQ ID NO: 175, SEQ IDNO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180,SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ IDNO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189,SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ IDNO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, or SEQ ID NO:198.

Aspects of the present specification disclose, in part, a Protein Ccleavage site as an inactivation cleavage site. As used herein, the term“Protein C cleavage site” refers to a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by Protein C underconditions suitable for Protein C protease activity. It is envisionedthat any amino acid sequence cleaved by Protein C can be useful inaspects of the present specification. Although exceptions are known, ageneralized consensus sequence for a Protein C cleavage site isX₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 199), where X₁ is preferentially abasic amino acid like K and R, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₂ is preferentially an acidic amino acidlike D and E, an amidic amino acid like Q and N, a basic amino acid likeK and R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₃ is preferentially an amidic amino acid like Q and N, a basicamino acid like K and R, an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ ispreferentially an amidic amino acid like Q and N, a basic amino acidlike K and R, an uncharged amino acid like C, S, and T, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₅ ispreferentially an amidic amino acid like Q and N, a basic amino acidlike K and R, an aromatic hydrophobic amino acid like F, W and Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ ispreferentially an amidic amino acid like Q and N, a positive amino acidlike H, K, and R, an uncharged amino acid like C, S, and T, or anaromatic hydrophobic amino acid like F, W and Y; X₇ is preferentially anacidic amino acid like D and E, an amidic amino acid like Q and N, abasic amino acid like K and R, an uncharged amino acid like C, S, and T,or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M.Table 4 lists exemplary reference cleavage sites for Protein C (SEQ IDNO: 200-209). Additional Protein C cleavage sites are well known in theart or can be defined by routine methods. See, e.g., L. Shen and B.Dahiback, Protein C. In Handbook of Proteolytic Enzymes, pp. 1673-1677(A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds; Elsevier,London, 2d, 2004); O. Schilling and C. M. Overall, Proteome-Derived,Database-Searchable Peptide Libraries for Identifying Protease CleavageSites, Nat. Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al.,MEROPS: The Peptidase Database, Nucleic Acids Res. 36(Database issue):D320-D325 (2008); Neil D. Rawlings, et al., MEROPS: The PeptidaseDatabase, Nucleic Acids Res. 38(Database issue): D227-D233 (2010); NeilD. Rawlings, et al., A Large and Accurate Collection of PeptidaseCleavages in the MEROPS Database, Database in press (2010), each ofwhich is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Protein C cleavage site. In an aspect of thisembodiment, a Protein C cleavage site comprises the consensus sequenceSEQ ID NO: 199, where X₁ is a basic amino acid like K and R, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₂ is anacidic amino acid like D and E, an amidic amino acid like Q and N, abasic amino acid like K and R, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₃ and X₄ are independently an amidicamino acid like Q and N, a basic amino acid like K and R, an unchargedamino acid like C, S, and T, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₅ is an amidic amino acid like Q and N,a basic amino acid like K and R, an aromatic hydrophobic amino acid likeF, W and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₆ is an amidic amino acid like Q and N, a positive amino acidlike H, K, and R, an uncharged amino acid like C, S, and T, or anaromatic hydrophobic amino acid like F, W and Y; X₇ is an acidic aminoacid like D and E, an amidic amino acid like Q and N, a basic amino acidlike K and R, an uncharged amino acid like C, S, and T, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M. In another aspectof this embodiment, a Protein C cleavage site comprises the sequence SEQID NO: 199, where X₁ is K, R, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₂ is D, E, Q, N, or K; X₃ is P, L, T, Q,K, or R; X₄ is G, I, S, N, or K; X₅ is Q, N, K, F, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ is F, S, N, Q,K, or H; X₇ is L, I, T, K, D, E, Q, or N. In other aspects of thisembodiment, a Protein C cleavage site comprises, e.g., SEQ ID NO: 200,SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ IDNO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO:209.

Aspects of the present specification disclose, in part, a Plasminogencleavage site as an inactivation cleavage site. As used herein, the term“Plasminogen cleavage site” refers to a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by Plasminogen underconditions suitable for Plasminogen protease activity. It is envisionedthat any amino acid sequence cleaved by Plasminogen can be useful inaspects of the present specification. Although exceptions are known, ageneralized consensus sequence for a Plasminogen cleavage site isX₁X₂X₃(R/K)*X₄X₅X₆X₇ (SEQ ID NO: 210), where X₁ is preferentially apositive amino acid like H, K and R, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is preferentially an amidic amino acid like N and Q, apositive amino acid like H, K and R, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₃ is preferentially an amidic amino acid like N and Q, anuncharged amino acid like C, S, and T, an aromatic amino acid like F, Wand Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₄ is preferentially a positive amino acid like H, K and R, anuncharged amino acid like C, S, and T, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₅ is preferentially a positiveamino acid like H, K and R, an uncharged amino acid like C, S, and T, oran aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ isany amino acid; X₇ is preferentially H, F, Y, R, an uncharged amino acidlike C, S, and T, an aliphatic hydrophobic amino acid like, G, P, A, V,L, I, and M. Table 4 lists exemplary reference cleavage sites forPlasminogen (SEQ ID NO: 211-240). Additional Plasminogen cleavage sitesare well known in the art or can be defined by routine methods. See,e.g., O. Schilling and C. M. Overall, Proteome-Derived,Database-Searchable Peptide Libraries for Identifying Protease CleavageSites, Nat. Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al.,MEROPS: The Peptidase Database, Nucleic Acids Res. 36(Database issue):D320-D325 (2008); Neil D. Rawlings, et al., MEROPS: The PeptidaseDatabase, Nucleic Acids Res. 38(Database issue): D227-D233 (2010); NeilD. Rawlings, et al., A Large and Accurate Collection of PeptidaseCleavages in the MEROPS Database, Database in press (2010), each ofwhich is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Plasminogen cleavage site. In an aspect of thisembodiment, a Plasminogen cleavage site comprises the consensus sequenceSEQ ID NO: 211, where X₁ is a positive amino acid like H, K and R, anuncharged amino acid like C, S, and T, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₂ is an amidic amino acid like Nand Q, a positive amino acid like H, K and R, an uncharged amino acidlike C, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M; X₃ is an amidic amino acid like N and Q, an unchargedamino acid like C, S, and T, an aromatic amino acid like F, W and Y, oran aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ isa positive amino acid like H, K and R, an uncharged amino acid like C,S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₅ is a positive amino acid like H, K and R, an uncharged aminoacid like C, S, and T, or an aliphatic hydrophobic amino acid like, G,P, A, V, L, I, and M; X₆ is any amino acid; X₇ is H, F, Y, R, anuncharged amino acid like C, S, and T, an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M. In another aspect of thisembodiment, a Plasminogen cleavage site comprises the sequence SEQ IDNO: 211, where X₁ is K, R, S, T, A, G, L, or P; X₂ is D, E, Q, N, K, R,S, T, A, G, I or L; X₃ is N, Q, S, F, Y, A or L; X₄ is K, R, S, A, G, L,or V; X₅ is K, R, N, S, F, Y, A, I, L, P, or V; X₆ is K, R, N, S, F, Y,A, G, L, P or V; X₇ is R, S, T, F, Y, A, G, I, L, or P. In other aspectsof this embodiment, a Plasminogen cleavage site comprises, e.g., SEQ IDNO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215,SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ IDNO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224,SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ IDNO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233,SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ IDNO: 238, SEQ ID NO: 239, or SEQ ID NO: 240.

Aspects of the present specification disclose, in part, a MatrixMetalloproteinase-2 cleavage site as an inactivation cleavage site. Asused herein, the term “Matrix Metalloproteinase-2 cleavage site” or“MMP-2 cleavage site” refers to a scissile bond together with adjacentor non-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by MMP-2 under conditions suitable forMMP-2 protease activity. It is envisioned that any amino acid sequencecleaved by MMP-2 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aMMP-2 cleavage site is X₁(P/A/V/L/I)X₂X₃*(V/L/I/F/Q)X₄X₅X₆ (SEQ ID NO:241), where X₁, X₂, X₃, X₄, X₅, and X₆ are any amino acid. Table 4 listsexemplary reference cleavage sites for MMP-2 (SEQ ID NO: 242-273).Additional MMP-2 cleavage sites are well known in the art or can bedefined by routine methods. See, e.g., O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Matrix Metalloproteinase-2 cleavage site. In anaspect of this embodiment, a Matrix Metalloproteinase-2 cleavage sitecomprises the consensus sequence SEQ ID NO: 241, where X₁, X₂, X₃, X₄,X₅, and X₆ are any amino acid. In another aspect of this embodiment, aMatrix Metalloproteinase-2 cleavage site comprises the consensussequence SEQ ID NO: 241, where X₁ is an acidic amino acid like D and E,an amidic amino acid like N and Q, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is an acidic amino acid like D and E, a basic amino acid likeK and R, an amidic amino acid like N and Q, an uncharged amino acid likeC, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₃ is H, an acidic amino acid like D and E, an amidic aminoacid like N and Q, an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ is abasic amino acid like K and R, an amidic amino acid like N and Q, anuncharged amino acid like C, S, and T, an aromatic amino acid like F, W,and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₅ is an acidic amino acid like D and E, a basic amino acid likeK and R, an amidic amino acid like N and Q, an uncharged amino acid likeC, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₆ is an acidic amino acid like D and E, a basic amino acidlike K and R, an amidic amino acid like N and Q, an uncharged amino acidlike C, S, and T, or an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M. In another aspect of this embodiment, a MatrixMetalloproteinase-2 cleavage site comprises the sequence SEQ ID NO: 241,where X₁ is G, P, A, V, L, I, S, T, E, or Q; X₂ is G, A, L, S, N, Q, W,or K; X₃ is G, P, A, S, Q, D, E, or H; X₄ is G, A, V, L, I, F, S, T, Q,or K; X₅ is G, A, V, S, T, Q, or K; X₆ is G, P, A, V, L, I, S, T, D, E,K, N, or Q. In another aspect of this embodiment, a MatrixMetalloproteinase-2 cleavage site comprises the sequence SEQ ID NO: 241,where X₁ is G, A, or L, or Q; X₂ is G, A, or S; X₃ is G, A, S, or N; X₄is A, V, L, I, or K; X₅ is G, A, or S; X₆ is G, P, A, V, L, or D. Inother aspects of this embodiment, a Matrix Metalloproteinase-2 cleavagesite comprises, e.g., SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244,SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ IDNO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253,SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ IDNO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262,SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ IDNO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271,SEQ ID NO: 272, or SEQ ID NO: 273.

Aspects of the present specification disclose, in part, a MatrixMetalloproteinase-9 cleavage site as an inactivation cleavage site. Asused herein, the term “Matrix Metalloproteinase-9 cleavage site” or“MMP-2 cleavage site” refers to a scissile bond together with adjacentor non-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by MMP-9 under conditions suitable forMMP-9 protease activity. It is envisioned that any amino acid sequencecleaved by MMP-9 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aMMP-9 cleavage site is X₁X₂X₃X₄*X₅X₆X₇X₈ (SEQ ID NO: 274), where X₁ ispreferentially F, an acidic amino acid like D and E, an amidic aminoacid like N and Q, a positive amino acid like H, K, and R, an unchargedamino acid like C, S, and T, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₂ is preferentially F, Y, S, T, anacidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₃ is preferentially F, Y, an acidicamino acid like D and E, an amidic amino acid like N and Q, a positiveamino acid like H, K, and R, an uncharged amino acid like C, S, and T,or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄is any amino acid; X₅ is preferentially S, T, an acidic amino acid likeD and E, an amidic amino acid like N and Q, a positive amino acid likeH, K, and R, an aromatic hydrophobic amino acid like F, W, and Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ isany amino acid; X₇ is any amino acid; X₈ is preferentially F, Y, anacidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M. Table 4 lists exemplary reference cleavage sites for MMP-9 (SEQID NO: 275-319). Additional MMP-9 cleavage sites are well known in theart or can be defined by routine methods. See, e.g., S. L. Kridel, etal., Substrate Hydrolysis by Matrix Metalloproteinase-9, J. Biol. Chem.276: 20572-20578 (2001); E. Y. Zhen, et al., Characterization ofMetalloprotease Cleavage Products of Human Articular Cartilage,Arthritis Rheum. 58: 2420-2431 (2008); O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Matrix Metalloproteinase-9 cleavage site. In anaspect of this embodiment, a Matrix Metalloproteinase-9 cleavage sitecomprises the consensus sequence SEQ ID NO: 274, where X₁ is F, anacidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is F, Y, S, T, an acidic amino acid like D and E, an amidicamino acid like N and Q, a positive amino acid like H, K, and R, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is F,Y, an acidic amino acid like D and E, an amidic amino acid like N and Q,a positive amino acid like H, K, and R, an uncharged amino acid like C,S, and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₄ is any amino acid; X₅ is S, T, an acidic amino acid like D andE, an amidic amino acid like N and Q, a positive amino acid like H, K,and R, an aromatic hydrophobic amino acid like F, W, and Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ isany amino acid; X₇ is any amino acid; X₈ is F, Y, an acidic amino acidlike D and E, an amidic amino acid like N and Q, a positive amino acidlike H, K, and R, an uncharged amino acid like C, S, and T, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M. Inanother aspect of this embodiment, a Matrix Metalloproteinase-9 cleavagesite comprises the consensus sequence SEQ ID NO: 274, where X₁ is G, V,L, I, F, S, Q, K, or R; X₂ is P, A, V, L, I, or S; X₃ is G, P, A, V, L,S, Q, E, K, or R; X₄ is G, P, A, V, L, F, S, N, E, or K; X₅ is A, V, L,I, M, F, S, Q, or K; X₆ is P, A, V, L, I, S, T, Q, E, K, or R; X₇ is G,A, V, L, S, or T; X₈ is G, P, A, V, L, F, T, D, E, K, or R. In anotheraspect of this embodiment, a Matrix Metalloproteinase-9 cleavage sitecomprises the consensus sequence SEQ ID NO: 274, where X₁ is G or L; X₂is P, A, or V; X₃ is P, A, R, K, or S; X₄ is G; X₅ is A, V, L, or I; X₆is T, Q, K, or R; X₇ is G, A, or S; X₈ is G, P, A, V, or E. In otheraspects of this embodiment, a Matrix Metalloproteinase-9 cleavage sitecomprises, e.g., SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ IDNO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282,SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ IDNO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291,SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ IDNO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300,SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ IDNO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309,SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ IDNO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318,or SEQ ID NO: 319.

Aspects of the present specification disclose, in part, a Furin cleavagesite as an inactivation cleavage site. As used herein, the term “Furincleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by Furin under conditions suitable forFurin protease activity. It is envisioned that any amino acid sequencecleaved by Furin can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aFurin cleavage site is (R/I/A)X₁(R/K/A/P)R*X₂*X₃X₄X₅ (SEQ ID NO: 320),where X₁, X₂, X₃, X₄, and X₅ are any amino acid. Table 4 lists exemplaryreference cleavage sites for Furin (SEQ ID NO: 321-346). AdditionalFurin cleavage sites are well known in the art or can be defined byroutine methods. See, e.g., A. Basak, et al., Implication of theProprotein Convertases Furin, PC5 And PC7 in the Cleavage of SurfaceGlycoproteins of Hong Kong, Ebola and Respiratory Syncytial Viruses: AComparative Analysis with Fluorogenic Peptides, Biochem. J. 353: 537-545(2001); O. Bader, et al., Processing of Predicted Substrates of FungalKex2 Proteinases from Candida albicans, C. glabrata, Saccharomycescerevisiae and Pichia pastoris, BMC Microbiol. 8: 116 (2008); O.Schilling and C. M. Overall, Proteome-Derived, Database-SearchablePeptide Libraries for Identifying Protease Cleavage Sites, Nat.Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: ThePeptidase Database, Nucleic Acids Res. 36(Database issue): D320-D325(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,Nucleic Acids Res. 38(Database issue): D227-D233 (2010); Neil D.Rawlings, et al., A Large and Accurate Collection of Peptidase Cleavagesin the MEROPS Database, Database in press (2010), each of which isincorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Furin cleavage site. In an aspect of thisembodiment, a Furin cleavage site comprises the consensus sequence SEQID NO: 320, where X₁, X₂, X₃, X₄, and X₅ are any amino acid. In anotheraspect of this embodiment, a Furin cleavage site comprises the consensussequence SEQ ID NO: 320, where X₁ is F, S, T, an acidic amino acid likeD and E, an amidic amino acid like N and Q, a positive amino acid likeH, K, and R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₂ is G, P, M, F, Y, an acidic amino acid like D and E, anamidic amino acid like N and Q, a positive amino acid like H, K, and R,or an uncharged amino acid like C, S, and T; X₃ is G, P, A, V, L, I, F,W, S, T, N, Q, D, H, K, or R; X₄ is F, Y, an acidic amino acid like Dand E, an amidic amino acid like N and Q, a positive amino acid like H,K, and R, an uncharged amino acid like C, S, and T, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₅ is F, Y, anacidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M. In another aspect of this embodiment, a Furin cleavage sitecomprises the consensus sequence SEQ ID NO: 320, where X₁ is K, R, S orT; X₂ is D, E, S, A or G; X₃ is A, V, L, or I; and X₄ is S, G, D, E orR; and X₅ is G, P, A, S, T, Q, D, or E. In other aspects of thisembodiment, a Furin cleavage site comprises, e.g., SEQ ID NO: 321, SEQID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO:326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO:335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO:344, SEQ ID NO: 345, or SEQ ID NO: 346.

Aspects of the present specification disclose, in part, a u-PlasminogenActivator cleavage site as an inactivation cleavage site. As usedherein, the term “u-Plasminogen Activator cleavage site” or “u-PAcleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by u-PA under conditions suitable foru-PA protease activity. It is envisioned that any amino acid sequencecleaved by u-Plasminogen Activator can be useful in aspects of thepresent specification. Although exceptions are known, a generalizedconsensus sequence for a u-PA cleavage site is X₁X₂X₃(R/K)*X₄*X₅X₆X₇(SEQ ID NO: 347), where X₁ is any amino acid;, X₂ is preferentially anuncharged amino acid like C, S, and T, an aromatic amino acid like F, W,and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₃ is preferentially an amidic amino acid like N and Q, anuncharged amino acid like C, S, and T, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₄ is any amino acid; X₅ ispreferentially a basic amino acid like K and R, an aromatic amino acidlike F, W, and Y, or an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M; X₆ is preferentially an uncharged amino acid like C, S,and T, an aromatic amino acid like F, W, and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₇ is anyamino acid. Table 4 lists exemplary reference cleavage sites for u-PA(SEQ ID NO: 347-368). Additional u-PA cleavage sites are well known inthe art or can be defined by routine methods. See, e.g., V. Ellis,u-Plasminogen Activator. In Handbook of Proteolytic Enzymes, pp.1677-1683 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds;Elsevier, London, 2d, 2004); O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a u-Plasminogen Activator cleavage site. In an aspectof this embodiment, a u-Plasminogen Activator cleavage site comprisesthe consensus sequence SEQ ID NO: 347, where X₁ is any amino acid;, X₂is an uncharged amino acid like C, S, and T, an aromatic amino acid likeF, W, and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₃ is an amidic amino acid like N and Q, an uncharged aminoacid like C, S, and T, or an aliphatic hydrophobic amino acid like, G,P, A, V, L, I, and M; X₄ is any amino acid; X₅ is a basic amino acidlike K and R, an aromatic amino acid like F, W, and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ is an unchargedamino acid like C, S, and T, an aromatic amino acid like F, W, and Y, oran aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; andX₇ is any amino acid. In another aspect of this embodiment, au-Plasminogen Activator cleavage site comprises the consensus sequenceSEQ ID NO: 347, where X₁ is P, A, L, S, T, C, N, or R; X₂ is G, P, L, Y,S, or T; X₃ is G, A, S, or N; and X₄ is G, A, V, I, Y, S, or R; X₅ is P,V, L, F, or R; X₆ is G, A, V, Y, S, or T; and X₇ is G, V, L, F, Y, N, orH. In another aspect of this embodiment, a u-Plasminogen Activatorcleavage site comprises the consensus sequence SEQ ID NO: 347, where X₁is P, A, L, S, T, C, N, or R; X₂ is G, Y, or S; X₃ is G or S; and X₄ isG, A, V, I, Y, S, or R; X₅ is V or R; X₆ is T or Y; and X₇ is G, V, L,F, Y, N, or H. In other aspects of this embodiment, a u-PlasminogenActivator cleavage site comprises, e.g., SEQ ID NO: 348, SEQ ID NO: 349,NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354,SEQ ID NO: 355, or SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO:363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, orSEQ ID NO: 368.

Aspects of the present specification disclose, in part, a t-PlasminogenActivator cleavage site as an inactivation cleavage site. As usedherein, the term “t-Plasminogen Activator cleavage site” or “t-PAcleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by t-PA under conditions suitable fort-PA protease activity. It is envisioned that any amino acid sequencecleaved by t-PA can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for at-PA cleavage site is X₁X₂X₃(R/K)*X₄*X₅X₆X₇ (SEQ ID NO: 369), where X₁,X₂, X₃, X₄, X₅, X₆, and X₇ are any amino acid. Table 4 lists exemplaryreference cleavage sites for t-PA (SEQ ID NO: 370-373). Additional t-PAcleavage sites are well known in the art or can be defined by routinemethods. See, e.g., H. R. Lijnen and D. Collen, t-Plasminogen Activator.In Handbook of Proteolytic Enzymes, pp. 1684-1689 (A. J. Barrett, N. D.Rawlings, and J. F. Woessner, eds; Elsevier, London, 2d, 2004); O.Schilling and C. M. Overall, Proteome-Derived, Database-SearchablePeptide Libraries for Identifying Protease Cleavage Sites, Nat.Biotechnol. 26: 685-694 (2008); Neil D. Rawlings, et al., MEROPS: ThePeptidase Database, Nucleic Acids Res. 36(Database issue): D320-D325(2008); Neil D. Rawlings, et al., MEROPS: The Peptidase Database,Nucleic Acids Res. 38(Database issue): D227-D233 (2010); Neil D.Rawlings, et al., A Large and Accurate Collection of Peptidase Cleavagesin the MEROPS Database, Database in press (2010), each of which isincorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a t-Plasminogen Activator cleavage site. In an aspectof this embodiment, a t-Plasminogen Activator cleavage site comprisesthe consensus sequence SEQ ID NO: 369, where X₁, X₂, X₃, X₄, X₅, X₆, andX₇ are any amino acid. In another aspect of this embodiment, at-Plasminogen Activator cleavage site comprises the consensus sequenceSEQ ID NO: 369, where X₁ is an amidic amino acid like N and Q, anuncharged amino acid like C, S, and T, or an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M; X₂ is an amidic amino acid like Nand Q, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₃ is an amidic amino acid like N and Q, an aromatic hydrophobicamino acid like F, W and Y, or an aliphatic hydrophobic amino acid like,G, P, A, V, L, I, and M; X₄ is an aromatic hydrophobic amino acid likeF, W and Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₅ is a basic amino acid like K and R, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₆ is an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₇ is anacidic amino acid like D and E, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M. In another aspect of this embodiment, at-Plasminogen Activator cleavage site comprises the consensus sequenceSEQ ID NO: 369, where X₁ is A, P, C, or N; X₂ is A, L, P, or Q; X₃ is G,L, S, or F; X₄ is I, V, M, or Y; X₅ is A, V, or K; X₆ is G, V, or P; andX₇ is G, L, or D. In other aspects of this embodiment, a t-PlasminogenActivator cleavage site comprises, e.g., SEQ ID NO: 370, SEQ ID NO: 371,SEQ ID NO: 372, or SEQ ID NO: 373.

Aspects of the present specification disclose, in part, a Tryptase-εcleavage site as an inactivation cleavage site. As used herein, the term“Tryptase-ε cleavage site” or “Prosemin cleavage site” refers to ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by Tryptase-ε under conditions suitable for Tryptase-ε proteaseactivity. It is envisioned that any amino acid sequence cleaved byTryptase-ε can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aTryptase-ε cleavage site is *(R/K)X₁X₂X₃X₄(D/E) (SEQ ID NO: 374), whereX₁, X₂, X₃, and X₄, are independently an aliphatic hydrophobic aminoacid like, G, P, A, V, L, I, and M. Table 4 lists exemplary referencecleavage sites for Tryptase-ε (SEQ ID NO: 375-386). AdditionalTryptase-ε cleavage sites are well known in the art or can be defined byroutine methods. See, e.g., O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Tryptase-E cleavage site. In an aspect of thisembodiment, a Tryptase-ε cleavage site comprises the consensus sequenceSEQ ID NO: 374, where X₁, X₂, X₃, and X₄, are independently an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M. In another aspectof this embodiment, a Tryptase-ε cleavage site comprises the consensussequence SEQ ID NO: 374, where X₁ is I or V; X₂ is I or V; X₃ is G or S;X₄ is G or S. In other aspects of this embodiment, a Tryptase-ε cleavagesite comprises, e.g., SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377,SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ IDNO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, or SEQ ID NO:386.

Aspects of the present specification disclose, in part, a Mouse MastCell Protease-7 cleavage site as an inactivation cleavage site. As usedherein, the term “Mouse Mast Cell Protease-7 cleavage site” or “mMCP-7cleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by mMCP-7 under conditions suitable formMCP-7 protease activity. It is envisioned that any amino acid sequencecleaved by mMCP-7 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for amMCP-7 cleavage site is X₁X₂X₃(K/R)*X₄X₅X₆X₇ (SEQ ID NO: 387), where X₁is any amino acid; X₂ is preferentially an amidic amino acid like N orQ, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M;X₃ is preferentially an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M; and X₄, X₅, X₆, X₇ are any amino acid. Table 4 listsexemplary reference cleavage sites for mMMCP-7 (SEQ ID NO: 388-391).Additional mMMCP-7 cleavage sites are well known in the art or can bedefined by routine methods. See, e.g., O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Mouse Mast Cell Protease-7 cleavage site. In anaspect of this embodiment, a Mouse Mast Cell Proteas-7 cleavage sitecomprises the consensus sequence SEQ ID NO: 387, where X₁ is any aminoacid; X₂ is an amidic amino acid like N or Q, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₄, X₅, X₆, X₇are independently any amino acid. In another aspect of this embodiment,a Mouse Mast Cell Protease-7 cleavage site comprises the consensussequence SEQ ID NO: 387, where X₁ is any amino acid; X₂ is G, S, or Q;X₃ is A, P or S; and X₄, X₅, X₆, X₇ are any amino acid. In other aspectsof this embodiment, a Mouse Mast Cell Protease-7 cleavage sitecomprises, e.g., SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, or SEQID NO: 391.

Aspects of the present specification disclose, in part, anEndothelin-Converting Enzyme-1 cleavage site as an inactivation cleavagesite. As used herein, the term “Endothelin-Converting Enzyme-1 cleavagesite” or “ECE-1 cleavage site” refers to a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by ECE-1 under conditionssuitable for ECE-1 protease activity. It is envisioned that any aminoacid sequence cleaved by ECE-1 can be useful in aspects of the presentspecification. Although exceptions are known, a generalized consensussequence for an ECE-1 cleavage site is X₁X₂X₃X₄*(F/L/I/V/Y)X₅X₆X₇ (SEQID NO: 392), where X₁, X₂, X₃, X₄, X₅, X₆, and X₇ are any amino acid.Table 4 lists exemplary reference cleavage sites for ECE-1 (SEQ ID NO:393-412). Additional ECE-1 cleavage sites are well known in the art orcan be defined by routine methods. See, e.g., K. Ahn and G. D. Johnson,Endothelin-Converting Enzyme-1. In Handbook of Proteolytic Enzymes, pp.429-434 (A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds;Elsevier, London, 2d, 2004); O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises an Endothelin-Converting Enzyme-1 cleavage site. Inan aspect of this embodiment, an Endothelin-Converting Enzyme-1 cleavagesite comprises the consensus sequence SEQ ID NO: 392, where X₁, X₂, X₃,X₄, X₅, X₆, and X₇ are independently any amino acid. In another aspectof this embodiment, an Endothelin-Converting Enzyme-1 cleavage sitecomprises the consensus sequence SEQ ID NO: 392, where X₁ is G, P, Y, anacidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, or an uncharged amino acid like C,S, and T; X₂ is F, an acidic amino acid like D and E, an amidic aminoacid like N and Q, a positive amino acid like H, K, and R, an unchargedamino acid like C, S, and T, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₃ is S, an acidic amino acid like D andE, an amidic amino acid like N and Q, a positive amino acid like H, K,and R, an aromatic hydrophobic amino acid like F, W and Y, or analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ is S,an acidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, an aromatic hydrophobic amino acidlike F, W and Y, or an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M; X₅ is F, W, S, C, N, E, a positive amino acid like H, K,and R, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₆ is G, P, V, L, F, Y, an acidic amino acid like D and E, anamidic amino acid like N and Q, a positive amino acid like H, K, and R,or an uncharged amino acid like C, S, and T; and X₇ is P, A, V, L, M, F,Y, S, N, D, or K. In another aspect of this embodiment, anEndothelin-Converting Enzyme-1 cleavage site comprises the consensussequence SEQ ID NO: 392, where X₁ is G, P, Y, C, D, K, R, or H; X₂ is P,L, I, F, S, C, Q, D, R, or H; X₃ is V, L, I, S, Q, K, or R; X₄ is G, P,L, F, Y, W, or R; X₅ is V, I, M, F, N, R, or H; X₆ is P, L, F, T, E, orH; and X₇ is P, V, L, F, S, N, D, or K. In another aspect of thisembodiment, an Endothelin-Converting Enzyme-1 cleavage site comprisesthe consensus sequence SEQ ID NO: 392, where X₁ is G, D, or H; X₂ is Ior F; X₃ is V, I, S, Q or K; X₄ is P, F, or W; X₅ is I, N, R, or H; X₆is L, T, or H; and X₇ is P, S, or D. In other aspects of thisembodiment, an Endothelin-Converting Enzyme-1 cleavage site comprises,e.g., SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396,SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ IDNO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405,SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ IDNO: 410, SEQ ID NO: 411, or SEQ ID NO: 412.

Aspects of the present specification disclose, in part, a Kellblood-group protein cleavage site as an inactivation cleavage site. Asused herein, the term “Kell blood-group protein cleavage site” or KBGPcleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by KBGP under conditions suitable forKBGP protease activity. It is envisioned that any amino acid sequencecleaved by KBGP can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aKBGP cleavage site is X₁X₂X₃X₄*X₅X₆X₇X₈ (SEQ ID NO: 413), where X₁ ispreferentially an acidic amino acid like D and E; X₂ is preferentiallyan aliphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ ispreferentially an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; X₄ is preferentially an aromatic amino acid like F, W, and Y;X₅ is preferentially an aliphatic hydrophobic amino acid like, G, P, A,V, L, I, and M; X₆ is preferentially an amidic amino acid like N and Q;X₇ is an uncharged amino acid like C, S, and T; X₈ is preferentially analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M. Table 4lists exemplary reference cleavage sites for KBGP (SEQ ID NO: 414-415).Additional KBGP cleavage sites are well known in the art or can bedefined by routine methods. See, e.g., O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Kell blood-group protein cleavage site. In anaspect of this embodiment, a Kell blood-group protein cleavage sitecomprises the consensus sequence SEQ ID NO: 413, where X₁ is an acidicamino acid like D and E; X₂ is T or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₃ is an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; X₄ is an aromatic amino acid like F, W,and Y; X₅ is T or an aliphatic hydrophobic amino acid like, G, P, A, V,L, I, and M; X₆ is an amidic amino acid like N and Q; X₇ is an unchargedamino acid like C, S, and T, or a C-beta branched amino acid like I, V,or T; X₈ is an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M. In another aspect of this embodiment, a Kell blood-group proteincleavage site comprises the consensus sequence SEQ ID NO: 413, where X₁is D; X₂ is I, V, or T; X₃ is I, V, or T; X₄ is W; X₅ is I, V, or T; X₆is N; X₇ is T; X₈ is P. In other aspects of this embodiment, a Kellblood-group protein cleavage site comprises, e.g., SEQ ID NO: 414 or SEQID NO: 415.

Aspects of the present specification disclose, in part, a Cathepsin Lcleavage site as an inactivation cleavage site. As used herein, the term“Cathepsin L cleavage site” refers to a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by Cathepsin L underconditions suitable for Cathepsin L protease activity. It is envisionedthat any amino acid sequence cleaved by Cathepsin L can be useful inaspects of the present specification. Although exceptions are known, ageneralized consensus sequence for a Cathepsin L cleavage site isX₁X₂X₃X₄*X₅X₆X₇X₈ (SEQ ID NO: 416), where X₁ is preferentially W, anacidic amino acid like D and E, an amidic amino acid like N and Q, apositive amino acid like H, K, and R, an uncharged amino acid like C, S,and T, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I,and M; X₂ is any amino acid; X₃ is preferentially L, V, F or Y; and X₄,X₅, X₆, X₇, and X₈ are any amino acid. Table 4 lists exemplary referencecleavage sites for Cathepsin L (SEQ ID NO: 417-443). AdditionalCathepsin L cleavage sites are well known in the art or can be definedby routine methods. See, e.g., J. C. Kelly, et al., Profiling of CalpainActivity with a Series of FRET-Based Substrates, Biochim. Biophys. Acta1794: 1505-1509 (2009); O. Schilling and C. M. Overall,Proteome-Derived, Database-Searchable Peptide Libraries for IdentifyingProtease Cleavage Sites, Nat. Biotechnol. 26: 685-694 (2008); Neil D.Rawlings, et al., MEROPS: The Peptidase Database, Nucleic Acids Res.36(Database issue): D320-D325 (2008); Neil D. Rawlings, et al., MEROPS:The Peptidase Database, Nucleic Acids Res. 38(Database issue): D227-D233(2010); Neil D. Rawlings, et al., A Large and Accurate Collection ofPeptidase Cleavages in the MEROPS Database, Database in press (2010),each of which is incorporated by reference in its entirety.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a Cathepsin L cleavage site. In an aspect of thisembodiment, a Cathepsin L cleavage site comprises the consensus sequenceSEQ ID NO: 416, where X₁ is W, an acidic amino acid like D and E, anamidic amino acid like N and Q, a positive amino acid like H, K, and R,an uncharged amino acid like C, S, and T, or an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; X₂ is any amino acid; X₃ is L,V, F or Y; and X₄, X₅, X₆, X₇, and X₈ are any amino acid. In anotheraspect of this embodiment, a Cathepsin L cleavage site comprises theconsensus sequence SEQ ID NO: 416, where X₁ is G, P, A, L, Q, E, or K;X₂ is an aromatic amino acid like F, W, and Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₃ is L, V, F orY; X₄ is G, A, F, T, Q, E, K, or R; X₅ is G, A, S, an acidic amino acidlike D and E, an amidic amino acid like N and Q, or a positive aminoacid like H, K, and R; X₆ is P, A, L, I, S, Q, an acidic amino acid likeD and E, or a positive amino acid like H, K, and R; X₇ is a positiveamino acid like H, K, and R, or an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; and X₈ is P, L, S, T, an acidic aminoacid like D and E, an amidic amino acid like N and Q, or a basic aminoacid like K, and R. In another aspect of this embodiment, a Cathepsin Lcleavage site comprises the consensus sequence SEQ ID NO: 416, where X₁is G, A, Q, E, or K; X₂ is G, P, L, or F; X₃ is L, V, F or Y; X₄ is G,A, F, T, Q, E, K, or R; X₅ is A, S, Q, E, K, or R; X₆ is P, A, L, I, S,or E; X₇, P, L, or R; and X₈ is P, L, S, or K. In other aspects of thisembodiment, a Cathepsin L cleavage site comprises, e.g., SEQ ID NO: 417,SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQ ID NO: 421, SEQ IDNO: 422, SEQ ID NO: 423, SEQ ID NO: 424, SEQ ID NO: 425, SEQ ID NO: 426,SEQ ID NO: 427, SEQ ID NO: 428, SEQ ID NO: 429, SEQ ID NO: 430, SEQ IDNO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435,SEQ ID NO: 436, SEQ ID NO: 437, SEQ ID NO: 438, SEQ ID NO: 439, SEQ IDNO: 440, SEQ ID NO: 441, SEQ ID NO: 442, or SEQ ID NO: 443.

Aspects of the present specification disclose, in part, a PAR1 cleavagesite as an inactivation cleavage site. As used herein, the term “PAR1cleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by PAR1 under conditions suitable forPAR1 protease activity. It is envisioned that any amino acid sequencecleaved by PAR1 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aPAR1 cleavage site is X₁X₂X₃X₄(K/R)X₅ (SEQ ID NO: 444), where X₁ ispreferentially a small non-polar amino acid like A, C G, S, and T; X₂ ispreferentially a large non-polar amino acid like F, I, L, M, V, or anaromatic amino acid like F, H, W, or Y; X₃ is preferentially a largenon-polar amino acid like F, I, L, M, V, or an aromatic amino acid likeF, H, W, or Y; X₄ is preferentially an aliphatic hydrophobic amino acidlike, G, P, A, V, L, I, and M; and X₅ is preferentially an amidic aminoacid like N and Q, or an aromatic hydrophobic amino acid like F, W, orY. Table 4 lists exemplary reference cleavage sites for PAR1 (SEQ ID NO:445-452). Additional PAR1 cleavage sites are well known in the art orcan be defined by routine methods.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a PAR1 cleavage site. In an aspect of thisembodiment, a PAR1 cleavage site comprises the consensus sequence SEQ IDNO: 444, where X₁ is a small non-polar amino acid like A, C G, S, and T;X₂ is a large non-polar amino acid like F, I, L, M, V, or an aromaticamino acid like F, H, W, or Y; X₃ is a large non-polar amino acid likeF, I, L, M, V, or an aromatic amino acid like F, H, W, or Y; X₄ is analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; and X₅is an amidic amino acid like N and Q, or an aromatic hydrophobic aminoacid like F, W, or Y. In another aspect of this embodiment, a PAR1cleavage site comprises the consensus sequence SEQ ID NO: 444, where X₁is S, T, or G; X₂ is F or Y; X₃ is L, P, or F; X₄ is A, G, I, or L; andX₅ is F or N. In other aspects of this embodiment, a PAR1 cleavage sitecomprises, e.g., SEQ ID NO: 445, SEQ ID NO: 446, SEQ ID NO: 447, SEQ IDNO: 448, SEQ ID NO: 449, SEQ ID NO: 450, SEQ ID NO: 451, or SEQ ID NO:452.

Aspects of the present specification disclose, in part, a PAR2 cleavagesite as an inactivation cleavage site. As used herein, the term “PAR2cleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by PAR2 under conditions suitable forPAR2 protease activity. It is envisioned that any amino acid sequencecleaved by PAR2 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aPAR2 cleavage site is X₁X₂X₃X₄(K/R)X₅ (SEQ ID NO: 453), where X₁ ispreferentially a small non-polar amino acid like A, C G, S, and T; X₂ ispreferentially a large non-polar amino acid like F, I, L, M, V; X₃ ispreferentially a large non-polar amino acid like F, I, L, M, V; X₄ ispreferentially an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; and X₅ is preferentially a large non-polar amino acid like F,I, L, M, V. Table 4 lists exemplary reference cleavage sites for PAR2(SEQ ID NO: 454-455). Additional PAR2 cleavage sites are well known inthe art or can be defined by routine methods.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a PAR2 cleavage site. In an aspect of thisembodiment, a PAR2 cleavage site comprises the consensus sequence SEQ IDNO: 453, where X₁ is a small non-polar amino acid like A, C G, S, and T;X₂ is a large non-polar amino acid like F, I, L, M, V; X₃ is a largenon-polar amino acid like F, I, L, M, V; X₄ is an aliphatic hydrophobicamino acid like, G, P, A, V, L, I, and M; and X₅ is a large non-polaramino acid like F, I, L, M, V. In another aspect of this embodiment, aPAR2 cleavage site comprises the consensus sequence SEQ ID NO: 453,where X₁ is S; X₂ is I or L; X₃ is I or L; X₄ is A or G; X₅ is L or V.In other aspects of this embodiment, a PAR2 cleavage site comprises,e.g., SEQ ID NO: 454 or SEQ ID NO: 455.

Aspects of the present specification disclose, in part, a PAR3 cleavagesite as an inactivation cleavage site. As used herein, the term “PAR3cleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by PAR3 under conditions suitable forPAR3 protease activity. It is envisioned that any amino acid sequencecleaved by PAR3 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aPAR3 cleavage site is X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 456), where X₁ ispreferentially a small non-polar amino acid like A, C G, S, and T; X₂ ispreferentially a large non-polar amino acid like F, I, L, M, V; X₃ ispreferentially an amidic amino acid like N and Q, or a basic amino acidlike K and R; X₄ is preferentially a small non-polar amino acid like A,C G, S, and T; X₅ is preferentially a small non-polar amino acid like A,C G, S, and T, or a small polar amino acid like D, N, or P; and X₆ ispreferentially an acidic amino acid like D and E, or a small polar aminoacid like D, N, or P. Table 4 lists exemplary reference cleavage sitesfor PAR3 (SEQ ID NO: 457-459). Additional PAR3 cleavage sites are wellknown in the art or can be defined by routine methods.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises a PAR3 cleavage site. In an aspect of thisembodiment, a PAR3 cleavage site comprises the consensus sequence SEQ IDNO: 456, where X₁ is a small non-polar amino acid like A, C G, S, and T;X₂ is a large non-polar amino acid like F, I, L, M, V; X₃ is an amidicamino acid like N and Q, or a basic amino acid like K and R; X₄ is asmall non-polar amino acid like A, C G, S, and T; X₅ is a smallnon-polar amino acid like A, C G, S, and T, or a small polar amino acidlike D, N, or P; and X₆ is an acidic amino acid like D and E, or a smallpolar amino acid like D, N, or P. In another aspect of this embodiment,a PAR3 cleavage site comprises the consensus sequence SEQ ID NO: 456,where X₁ is S or T; X₂ is F; X₃ is N or R; X₄ is A or G; X₅ is A, G, orN and X₆ is P or E. In other aspects of this embodiment, a PAR3 cleavagesite comprises, e.g., SEQ ID NO: 457, SEQ ID NO: 458, or SEQ ID NO: 459.

Aspects of the present specification disclose, in part, a PAR4 cleavagesite as an inactivation cleavage site. As used herein, the term “PAR4cleavage site” refers to a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by PAR4 under conditions suitable forPAR4 protease activity. It is envisioned that any amino acid sequencecleaved by PAR4 can be useful in aspects of the present specification.Although exceptions are known, a generalized consensus sequence for aPAR4 cleavage site is X₁X₂X₃X₄(K/R/Q/F)X₅ (SEQ ID NO: 460), where X₁ ispreferentially a small non-polar amino acid like A, C G, S, and T; X₂ ispreferentially a large non-polar amino acid like F, I, L, M, V, or anaromatic amino acid like F, H, W, or Y; X₃ is preferentially analiphatic hydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ ispreferentially an aliphatic hydrophobic amino acid like, G, P, A, V, L,I, and M; and X₅ is preferentially a basic amino acid like K and R, anaromatic hydrophobic amino acid like F, W, or Y, or an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M. Table 4 listsexemplary reference cleavage sites for PAR4 (SEQ ID NO: 461-478).Additional PAR4 cleavage sites are well known in the art or can bedefined by routine methods. Thus, in an embodiment, a Clostridial toxinor Clostridial toxin chimeric comprises a PAR4 cleavage site. In anaspect of this embodiment, a PAR4 cleavage site comprises the consensussequence SEQ ID NO: 460, where X₁ is a small non-polar amino acid likeA, C G, S, and T; X₂ is a large non-polar amino acid like F, I, L, M, V,or an aromatic amino acid like F, H, W, or Y; X₃ is an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; X₄ is an aliphatichydrophobic amino acid like, G, P, A, V, L, I, and M; and X₅ is a basicamino acid like K and R, an aromatic hydrophobic amino acid like F, W,or Y, or an aliphatic hydrophobic amino acid like, G, P, A, V, L, I, andM. In another aspect of this embodiment, a PAR4 cleavage site comprisesthe consensus sequence SEQ ID NO: 460, where X₁ is A, G, S, or T; X₂ isF or Y; X₃ is A or P; X₄ is A or G; and X₅ is A, V, P, F, W, Y, or K. Inother aspects of this embodiment, a PAR4 cleavage site comprises, e.g.,SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ IDNO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469,SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ IDNO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, or SEQ ID NO:478.

The location of an inactivation cleavage site is a critical aspect thatis governed by several criteria. First, the placement of theinactivation cleavage site should not substantially affect the abilityof a Clostridial toxin or Clostridial toxin chimeric to intoxicate itstarget cell. As used herein, the term “not substantially affect,” withregards to intoxication, refers to a Clostridial toxin or Clostridialtoxin chimeric disclosed in the present specification that can stillexecute the overall intoxication mechanism whereby a Clostridial toxinor Clostridial toxin chimeric enters a target cell and proteolyticallycleaves a target substrate and encompasses the binding of a Clostridialtoxin or Clostridial toxin chimeric to a low or high affinity receptorcomplex, the internalization of the toxin/receptor complex, thetranslocation of the light chain into the cytoplasm and the enzymaticmodification of a target substrate.

In an aspect of this embodiment, a Clostridial toxin or Clostridialtoxin chimeric comprising an inactivation cleavage site can intoxicate atarget cell to the same extent as the same or similar Clostridial toxinor Clostridial toxin chimeric, but without the inactivation cleavagesite modification. In other aspects of this embodiment, a Clostridialtoxin or Clostridial toxin chimeric comprising an inactivation cleavagesite can intoxicate a target cell by, e.g., at least 50%, 60%, 70%, 80%,90% or 95% the extent as the same or similar Clostridial toxin orClostridial toxin chimeric, but without the inactivation cleavage sitemodification. In other aspects of this embodiment, a Clostridial toxinor Clostridial toxin chimeric comprising an inactivation cleavage sitecan intoxicate a target cell by, e.g., at most 50%, 60%, 70%, 80%, 90%or 95% the extent as the same or similar Clostridial toxin orClostridial toxin chimeric, but without the inactivation cleavage sitemodification.

Second, the placement of an inactivation cleavage site should be at asurface exposed region of the toxin or Clostridial toxin chimeric andnot buried internally within the protein or masked by secondarystructure elements. Proper surface exposure of the inactivation cleavagesite facilitates proper access of the site to its correspondingprotease, thereby enabling proteolytic cleavage. Proteolytic cleavage ofthe inactivation cleavage site by its corresponding proteasesubstantially inactivates the ability of the Clostridial toxin orClostridial toxin chimeric to intoxicate the cell. As used herein, theterm “substantially inactivates,” with regards to intoxication, refersto a Clostridial toxin or Clostridial toxin chimeric disclosed in thepresent specification that, after cleavage at an inactivation cleavagesite, has a reduced ability to execute the overall intoxicationmechanism whereby a Clostridial toxin or Clostridial toxin chimericenters a target cell and proteolytically cleaves a target substrate andencompasses the binding of a Clostridial toxin or Clostridial toxinchimeric to a low or high affinity receptor complex, the internalizationof the toxin/receptor complex, the translocation of the light chain intothe cytoplasm and the enzymatic modification of a target substrate.

In one aspect of this embodiment, proteolytic cleavage of a Clostridialtoxin or Clostridial toxin chimeric disclosed in the presentspecification at an inactivation cleavage site results in completeinability of the toxin to intoxicate a target cell as compared to thesame or similar Clostridial toxin or Clostridial toxin chimeric, but ina proteolytic uncleaved state (i.e., the intoxication cleavage site isintact or uncleaved). In other aspects of this embodiment, proteolyticcleavage of a Clostridial toxin or Clostridial toxin chimeric disclosedin the present specification at an inactivation cleavage site resultsin, e.g., at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%decreased ability to intoxicate a target cell as compared to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but in aproteolytic uncleaved state. In other aspects of this embodiment,proteolytic cleavage of a Clostridial toxin or Clostridial toxinchimeric disclosed in the present specification at an inactivationcleavage site results in, e.g., at most a 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 95% decreased ability to intoxicate a target cell ascompared to the same or similar Clostridial toxin or Clostridial toxinchimeric, but in a proteolytic uncleaved state.

In an aspect of the present specification, an inactivation cleavage siteis located within an inactivation cleavage site region. As used herein,the term “inactivation cleavage site region” refers to an amino acidsequence of a Clostridial toxin or Clostridial toxin chimeric that canbe modified to contain an inactivation cleavage site because suchmodification will not substantially disrupt the ability of the proteinto intoxicate a target cell; and upon exposure to its cognate protease,the inactivation cleavage site will be cleaved and substantiallyinactivate the Clostridial toxin or Clostridial toxin chimeric. Thelocation of an inactivation cleavage site can be anywhere within theinactivation cleavage site region, with the proviso that such locationwill not substantially affect the ability of the Clostridial toxin orClostridial toxin chimeric to intoxicate a target cell; and uponexposure to its cognate protease, cleavage of the inactivation cleavagesite will substantially inactivate the Clostridial toxin or Clostridialtoxin chimeric. Table 5 lists exemplary inactivation cleavage siteregions suitable for use with a Clostridial toxin or Clostridial toxinchimeric disclosed in the present specification.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric disclosed in the present specification comprises aninactivation cleavage site located within inactivation cleavage siteregion. In aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site located within inactivationcleavage site region from the translocation domain or the H_(CN)subdomain.

In other aspects of this embodiment, a Clostridial toxin or Clostridialtoxin chimeric disclosed in the present specification comprises aninactivation cleavage site is located within an inactivation cleavagesite region comprising amino acids 462-496 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids 458-492 of SEQ ID NO: 3;amino acids 464-487 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or SEQ ID NO: 10; amino acids 463-496 of SEQ ID NO: 11 or SEQ IDNO: 12; amino acids 458-491 of SEQ ID NO: 13 or SEQ ID NO: 14; aminoacids 434-467 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; aminoacids 453-486 of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; aminoacids 458-491 of SEQ ID NO: 21; amino acids 475-508 of SEQ ID NO: 22;amino acids 443-476 of SEQ ID NO: 23; or amino acids 434-467 of SEQ IDNO: 24 or SEQ ID NO: 25.

In yet other aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site is located within aninactivation cleavage site region comprising amino acids 618-634 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids614-630 of SEQ ID NO: 3; amino acids 605-621 of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino acids 613-629 ofSEQ ID NO: 11 or SEQ ID NO: 12; amino acids 609-625 of SEQ ID NO: 13 orSEQ ID NO: 14; amino acids 587-603 of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17; amino acids 604-620 of SEQ ID NO: 18; amino acids 605-621of SEQ ID NO: 19 or SEQ ID NO: 20; amino acids 610-626 of SEQ ID NO: 21;amino acids 627-643 of SEQ ID NO: 22; amino acids 596-612 of SEQ ID NO:23; or amino acids 587-603 of SEQ ID NO: 24 or SEQ ID NO: 25.

TABLE 5 Inactivation Cleavage Site Regions of Clostridial Toxins SEQInactivation Cleavage Site Regions Toxin ID NO: 1 2 3 4 5 6 7 8 BoNT/A 1L462-L496 T618-I634 G638-D651 L665-N687 N752-N765 N826-D835 T844-L863K871-A895 BoNT/B 2 L464-P487 A605-V621 G625-N638 L652-N674 N739-D752N813-A824 Y831-I850 S858-G882 BoNT/C1 3 L463-S496 I613-I629 G633-N646L660-E682 K747-Q760 H821-D830 S839-K858 N866-N890 BoNT/D 4 L458-S491I609-I625 G629-N642 L656-E678 K743-Q756 H817-D826 S835-K854 N862-N886BoNT/E 5 L434-D467 A587-V603 G607-N620 L634-N659 N724-D739 H800-Q809T818-I837 K845-D869 BoNT/F 6 L453-N486 A605-V621 G625-N638 L652-N677N742-N757 H818-N827 T836-I855 K863-G887 BoNT/G 7 L458-S491 S610-I626G630-N643 M657-N679 N744-D757 N818-N827 H836-I855 S863-G887 TeNT 8L475-S508 S627-V643 G647-N660 L674-Q696 K761-E774 N835-K844 V854-V871V879-N903 BaNT 9 L443-N476 A596-V612 G616-N629 L643-S668 N733-N748N809-P819 T828-I847 K855-G879 BuNT 10 L434-D467 A587-V603 G607-N620L634-S659 N724-D739 H800-Q809 T818-I837 K845-D869

In still other aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site is located within aninactivation cleavage site region comprising amino acids 638-651 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids634-647 of SEQ ID NO: 3; amino acids 625-638 of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino acids 633-646 ofSEQ ID NO: 11 or SEQ ID NO: 12; amino acids 629-642 of SEQ ID NO: 13 orSEQ ID NO: 14; amino acids 607-620 of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17; amino acids 624-637 of SEQ ID NO: 18; amino acids 625-638of SEQ ID NO: 19 or SEQ ID NO: 20; amino acids 630-643 of SEQ ID NO: 21;amino acids 647-660 of SEQ ID NO: 22; amino acids 616-629 of SEQ ID NO:23; or amino acids 607-620 of SEQ ID NO: 24 or SEQ ID NO: 25.

In further aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site is located within aninactivation cleavage site region comprising amino acids 665-687 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids661-683 of SEQ ID NO: 3; amino acids 652-674 of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino acids 660-682 ofSEQ ID NO: 11 or SEQ ID NO: 12; amino acids 656-678 of SEQ ID NO: 13 orSEQ ID NO: 14; amino acids 634-659 of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17; amino acids 651-676 of SEQ ID NO: 18; amino acids 652-677of SEQ ID NO: 19 or SEQ ID NO: 20; amino acids 657-679 of SEQ ID NO: 21;amino acids 674-696 of SEQ ID NO: 22; amino acids 643-668 of SEQ ID NO:23; or amino acids 634-659 of SEQ ID NO: 24 or SEQ ID NO: 25.

In other aspects of this embodiment, a Clostridial toxin or Clostridialtoxin chimeric disclosed in the present specification comprises aninactivation cleavage site is located within an inactivation cleavagesite region comprising amino acids 752-765 of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids 748-761 of SEQ ID NO: 3;amino acids 739-752 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, or SEQ ID NO: 10; amino acids 747-760 of SEQ ID NO: 11 or SEQ IDNO: 12; amino acids 743-756 of SEQ ID NO: 13 or SEQ ID NO: 14; aminoacids 724-739 of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; aminoacids 741-756 of SEQ ID NO: 18; amino acids 742-757 of SEQ ID NO: 19 orSEQ ID NO: 20; amino acids 744-757 of SEQ ID NO: 21; amino acids 761-774of SEQ ID NO: 22; amino acids 733-748 of SEQ ID NO: 23; or amino acids724-739 of SEQ ID NO: 24 or SEQ ID NO: 25.

In yet other aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site is located within aninactivation cleavage site region comprising amino acids 826-835 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids824-831 of SEQ ID NO: 3; amino acids 813-824 of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino acids 821-830 ofSEQ ID NO: 11 or SEQ ID NO: 12; amino acids 817-826 of SEQ ID NO: 13 orSEQ ID NO: 14; amino acids 800-809 of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17; amino acids 817-826 of SEQ ID NO: 18; amino acids 818-827of SEQ ID NO: 19 or SEQ ID NO: 20; amino acids 818-827 of SEQ ID NO: 21;amino acids 835-844 of SEQ ID NO: 22; amino acids 809-819 of SEQ ID NO:23; or amino acids 800-809 of SEQ ID NO: 24 or SEQ ID NO: 25.

In still other aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site is located within aninactivation cleavage site region comprising amino acids 844-863 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids840-859 of SEQ ID NO: 3;; amino acids 831-850 of SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino acids 839-858of SEQ ID NO: 11 or SEQ ID NO: 12; amino acids 835-854 of SEQ ID NO: 13or SEQ ID NO: 14; amino acids 818-837 of SEQ ID NO: 15, SEQ ID NO: 16,or SEQ ID NO: 17; amino acids 835-854 of SEQ ID NO: 18; amino acids836-855 of SEQ ID NO: 19 or SEQ ID NO: 20; amino acids 836-855 of SEQ IDNO: 21; amino acids 854-871 of SEQ ID NO: 22; amino acids 828-847 of SEQID NO: 23; or amino acids 818-837 of SEQ ID NO: 24 or SEQ ID NO: 25.

In further aspects of this embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationcomprises an inactivation cleavage site is located within aninactivation cleavage site region comprising amino acids 871-895 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; amino acids867-891 of SEQ ID NO: 3; amino acids 858-882 of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; amino acids 866-890 ofSEQ ID NO: 11 or SEQ ID NO: 12; amino acids 862-886 of SEQ ID NO: 13 orSEQ ID NO: 14; amino acids 845-869 of SEQ ID NO: 15, SEQ ID NO: 16, orSEQ ID NO: 17; amino acids 862-886 of SEQ ID NO: 18; amino acids 863-887of SEQ ID NO: 19 or SEQ ID NO: 20; amino acids 863-887 of SEQ ID NO: 21;amino acids 879-903 of SEQ ID NO: 22; amino acids 855-879 of SEQ ID NO:23; or amino acids 845-869 of SEQ ID NO: 24 or SEQ ID NO: 25.

In another aspect of this embodiment, a BoNT/A or BoNT/A chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BoNT/A or BoNT/A chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 462-496,618-634, 638-651, 665-687, 752-765, 826-835, 844-863, or 871-895 of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; or comprisingamino acids 458-492, 614-630, 634-647, 665-687, 748-761, 822-831,840-859, or 867-891 of SEQ ID NO: 3. In yet other aspects of thisembodiment, a BoNT/A comprising an inactivation cleavage site locatedwithin inactivation cleavage site region is encoded by SEQ ID NO: 530,SEQ ID NO: 532, SEQ ID NO: 534, or SEQ ID NO: 536. In still otheraspects of this embodiment, a BoNT/A comprising an inactivation cleavagesite located within inactivation cleavage site region comprises SEQ IDNO: 531, SEQ ID NO: 533, SEQ ID NO: 535, or SEQ ID NO: 537.

In yet another aspect of this embodiment, a BoNT/B or BoNT/B chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BoNT/B or BoNT/B chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 464-487,605-621, 625-638, 652-674, 739-752, 813-824, 831-850, or 858-882 of SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

In still another aspect of this embodiment, a BoNT/C1 or BoNT/C1chimeric disclosed in the present specification comprises aninactivation cleavage site is located within an inactivation cleavagesite region from the translocation domain or the H_(CN) subdomain. Inother aspects of this embodiment, a BoNT/C1 or BoNT/C1 chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regioncomprising amino acids 463-496, 613-629, 633-646, 660-682, 747-760,821-830, 839-858, or 866-890 of SEQ ID NO: 11 or SEQ ID NO: 12.

In a further aspect of this embodiment, a BoNT/D or BoNT/D chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BoNT/D or BoNT/D chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 458-491,609-625, 629-642, 656-678, 743-756, 817-826, 835-854, or 862-886 of SEQID NO: 13 or SEQ ID NO: 14.

In another aspect of this embodiment, a BoNT/E or BoNT/E chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BoNT/E or BoNT/E chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 434-467,587-603, 607-620, 634-659, 724-739, 800-809, 818-837, or 845-869 of SEQID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

In still another aspect of this embodiment, a BoNT/F or BoNT/F chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BoNT/F or BoNT/F chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 453-486,604-620, 624-637, 651-676, 741-756, 817-826, 835-854, or 862-886 of SEQID NO: 18; or comprising amino acids 453-486, 605-621, 625-638, 652-677,742-757, 818-827, 836-855, or 863-887 of SEQ ID NO: 19 or SEQ ID NO: 20.

In a further aspect of this embodiment, a BoNT/G or BoNT/G chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BoNT/G or BoNT/G chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 458-491,610-626, 630-643, 657-679, 744-757, 818-827, 836-855, or 863-887 of SEQID NO: 21.

In another aspect of this embodiment, a TeNT or TeNT chimeric disclosedin the present specification comprises an inactivation cleavage site islocated within an inactivation cleavage site region from thetranslocation domain or the H_(CN) subdomain. In other aspects of thisembodiment, a TeNT or TeNT chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 475-508,627-643, 647-660, 674-696, 761-774, 835-844, 854-871, or 879-903 of SEQID NO: 22.

In yet another aspect of this embodiment, a BaNT or BaNT chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BaNT or BaNT chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 443-476,596-612, 616-629, 643-668, 733-748, 809-819, 828-847, or 855-879 of SEQID NO: 23.

In still another aspect of this embodiment, a BuNT or BuNT chimericdisclosed in the present specification comprises an inactivationcleavage site is located within an inactivation cleavage site regionfrom the translocation domain or the H_(CN) subdomain. In other aspectsof this embodiment, a BuNT or BuNT chimeric disclosed in the presentspecification comprises an inactivation cleavage site is located withinan inactivation cleavage site region comprising amino acids 434-467,587-603, 607-620, 634-659, 724-739, 800-809, 818-837, or 845-869 of SEQID NO: 24 or SEQ ID NO: 25.

In an aspect of the present specification, a Clostridial toxin orClostridial toxin chimeric comprising an inactivation cleavage site hasa safety margin greater than the safety margin for the same or similarClostridial toxin or Clostridial toxin chimeric, but without theinactivation cleavage site. In other words, the addition of aninactivation cleavage site increases the safety margin of theClostridial toxin or Clostridial toxin chimeric relative to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but without theadditional inactivation cleavage site.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric comprising an inactivation cleavage site has a safety marginthat is greater relative to the same or similar Clostridial toxin orClostridial toxin chimeric, but without the inactivation cleavage site.In aspects of this embodiment, a Clostridial toxin or Clostridial toxinchimeric comprising an inactivation cleavage site has a safety marginthat is greater than, e.g., at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, 110%, at least 120%, at least 130%, at least140%, at least 150%, at least 160%, at least 170%, at least 180%, atleast 190%, at least 200%, 210%, at least 220%, at least 230%, at least240%, at least 250%, at least 260%, at least 270%, at least 280%, atleast 290%, or at least 300%, relative to the same or similarClostridial toxin or Clostridial toxin chimeric, but without theinactivation cleavage site. In other aspects of this embodiment, aClostridial toxin or Clostridial toxin chimeric comprising aninactivation cleavage site has a safety margin that is greater than,e.g., at most 10%, at most 20%, at most 30%, at most 40%, at most 50%,at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, 110%,at most 120%, at most 130%, at most 140%, at most 150%, at most 160%, atmost 170%, at most 180%, at most 190%, at most 200%, 210%, at most 220%,at most 230%, at most 240%, at most 250%, at most 260%, at most 270%, atmost 280%, at most 290%, or at most 300%, relative to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but without theinactivation cleavage site. In yet other aspects of this embodiment, aClostridial toxin or Clostridial toxin chimeric comprising aninactivation cleavage site has a safety margin that is greater by, e.g.,about 10% to about 300%, about 20% to about 300%, about 30% to about300%, about 40% to about 300%, about 50% to about 300%, about 60% toabout 300%, about 70% to about 300%, about 80% to about 300%, about 90%to about 300%, or about 100% to about 300%, relative to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but without theinactivation cleavage site.

In other aspects embodiment, a Clostridial toxin or Clostridial toxinchimeric comprising an inactivation cleavage site has a safety marginthat is greater than, e.g., at least 1-fold, at least 1-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, relativeto the same or similar Clostridial toxin or Clostridial toxin chimeric,but without the inactivation cleavage site. In yet other aspectsembodiment, a Clostridial toxin or Clostridial toxin chimeric comprisingan inactivation cleavage site has a safety margin that is greater than,e.g., at least 1-fold, at most 1-fold, at most 3-fold, at most 4-fold,at most 5-fold, at most 6-fold, at most 7-fold, at most 8-fold, at most9-fold, or at most 10-fold, relative to the same or similar Clostridialtoxin or Clostridial toxin chimeric, but without the inactivationcleavage site. In still other aspects of this embodiment, a Clostridialtoxin or Clostridial toxin chimeric comprising an inactivation cleavagesite has a safety margin that is greater by, e.g., about 1-fold to about10-fold, about 1-fold to about 9-fold, about 1-fold to about 8-fold,about 1-fold to about 7-fold, about 1-fold to about 6-fold, about 1-foldto about 5-fold, about 2-fold to about 10-fold, about 2-fold to about9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold,about 2-fold to about 6-fold, or about 2-fold to about 5-fold.

In another embodiment, a Clostridial toxin or Clostridial toxin chimericcomprises the addition of an inactivation cleavage site that increasesthe safety margin of the Clostridial toxin or Clostridial toxin chimericrelative to the same or similar Clostridial toxin or Clostridial toxinchimeric, but without the additional inactivation cleavage site. Inaspects of this embodiment, a Clostridial toxin or Clostridial toxinchimeric comprises the addition of an inactivation cleavage site thatincreases the safety margin of the Clostridial toxin or Clostridialtoxin chimeric relative to the same or similar Clostridial toxin orClostridial toxin chimeric, but without the additional inactivationcleavage site by, e.g., at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, 110%, at least 120%, at least 130%, at least140%, at least 150%, at least 160%, at least 170%, at least 180%, atleast 190%, at least 200%, 210%, at least 220%, at least 230%, at least240%, at least 250%, at least 260%, at least 270%, at least 280%, atleast 290%, or at least 300%. In other aspects of this embodiment, aClostridial toxin or Clostridial toxin chimeric comprises the additionof an inactivation cleavage site that increases the safety margin of theClostridial toxin or Clostridial toxin chimeric relative to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but without theadditional inactivation cleavage site by, e.g., at most 10%, at most20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, atmost 80%, at most 90%, at most 100%, 110%, at most 120%, at most 130%,at most 140%, at most 150%, at most 160%, at most 170%, at most 180%, atmost 190%, at most 200%, 210%, at most 220%, at most 230%, at most 240%,at most 250%, at most 260%, at most 270%, at most 280%, at most 290%, orat most 300%. In yet other aspects of this embodiment, a Clostridialtoxin or Clostridial toxin chimeric comprises the addition of aninactivation cleavage site that increases the safety margin of theClostridial toxin or Clostridial toxin chimeric relative to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but without theadditional inactivation cleavage site by, e.g., about 10% to about 300%,about 20% to about 300%, about 30% to about 300%, about 40% to about300%, about 50% to about 300%, about 60% to about 300%, about 70% toabout 300%, about 80% to about 300%, about 90% to about 300%, or about100% to about 300%.

In other aspects of this embodiment, a Clostridial toxin or Clostridialtoxin chimeric comprises the addition of an inactivation cleavage sitethat increases the safety margin of the Clostridial toxin or Clostridialtoxin chimeric relative to the same or similar Clostridial toxin orClostridial toxin chimeric, but without the additional inactivationcleavage site by, e.g., at least 1-fold, at least 1-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In yetother aspects of this embodiment, a Clostridial toxin or Clostridialtoxin chimeric comprises the addition of an inactivation cleavage sitethat increases the safety margin of the Clostridial toxin or Clostridialtoxin chimeric relative to the same or similar Clostridial toxin orClostridial toxin chimeric, but without the additional inactivationcleavage site by, e.g., at most 1-fold, at most 3-fold, at most 4-fold,at most 5-fold, at most 6-fold, at most 7-fold, at most 8-fold, at most9-fold, or at most 10-fold. In still other aspects of this embodiment, aClostridial toxin or Clostridial toxin chimeric comprises the additionof an inactivation cleavage site that increases the safety margin of theClostridial toxin or Clostridial toxin chimeric relative to the same orsimilar Clostridial toxin or Clostridial toxin chimeric, but without theadditional inactivation cleavage site by, e.g., about 1-fold to about10-fold, about 1-fold to about 9-fold, about 1-fold to about 8-fold,about 1-fold to about 7-fold, about 1-fold to about 6-fold, about 1-foldto about 5-fold, about 2-fold to about 10-fold, about 2-fold to about9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold,about 2-fold to about 6-fold, or about 2-fold to about 5-fold.

In another embodiment, an inactivation cleavage site region can bemodified to include a single inactivation cleavage site. In yet anotherembodiment, an inactivation cleavage site region can be modified toinclude a plurality inactivation cleavage site cleavage sites. Inaspects of this embodiment, an inactivation cleavage site cleavage siteregion can comprise, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10inactivation cleavage sites. In other aspects of this embodiment, aninactivation cleavage site cleavage site region can comprise, e.g., atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 inactivation cleavage sites. Inyet other aspects of this embodiment, an inactivation cleavage sitecleavage site region can comprise, e.g., 2-10 inactivation cleavagesites, 2-8 inactivation cleavage sites, 2-6 inactivation cleavage sites,2-4 inactivation cleavage sites, 2-3 inactivation cleavage sites, 3-9inactivation cleavage sites, 3-7 inactivation cleavage sites, 3-5inactivation cleavage sites, or 3-4 inactivation cleavage sites.

In another embodiment, an inactivation cleavage site region can bemodified to include only one type of inactivation cleavage site, suchas, e.g., a thrombin cleavage site. In still another embodiment, aninactivation cleavage site region can be modified to include a pluralityof different types of inactivation cleavage sites, such as, e.g., athrombin cleavage site, a Factor Xa cleavage site, MMP-2 cleavage site,and a MMP-9 cleavage site. In aspects of this embodiment, aninactivation cleavage site region can comprise, e.g., at least 2, 3, 4,or 5 different types of inactivation cleavage sites. In other aspects ofthis embodiment, an inactivation cleavage site region can comprise,e.g., at most 2, 3, 4, or 5 different types of inactivation cleavagesites. In other aspects of this embodiment, an inactivation cleavagesite region can comprise, e.g., 2-5 different types of inactivationcleavage sites, 2-4 different types of inactivation cleavage sites, 2-3different types of inactivation cleavage sites, 3-5 different types ofinactivation cleavage sites, or 3-4 different types of inactivationcleavage sites.

Modification of an inactivation cleavage site region to include ainactivation cleavage site can be accomplished by altering at least oneof the amino acids within the inactivation cleavage site region.Non-limiting examples of an amino acid alteration include a deletion ofan amino acid, an addition of an amino acid, or a substitution of anoriginal amino acid with a different amino acid. In aspects of thisembodiment, an inactivation cleavage site region is modified to includean inactivation cleavage site by altering, e.g., at least 1, 2, 3, 4, or5 amino acids within the inactivation cleavage site region. In otheraspects of this embodiment, an inactivation cleavage site region ismodified to include an inactivation cleavage site by altering, e.g., atmost 1, 2, 3, 4, or 5 amino acids within the inactivation cleavage siteregion. In yet aspects of this embodiment, an inactivation cleavage siteregion is modified to include an inactivation cleavage site by altering,e.g., 1-5 amino acids within the inactivation cleavage site region, 1-4amino acids within the inactivation cleavage site region, 1-3 aminoacids within the inactivation cleavage site region, 1-2 amino acidswithin the inactivation cleavage site region, 2-5 amino acids within theinactivation cleavage site region, 2-4 amino acids within theinactivation cleavage site region, 2-3 amino acids within theinactivation cleavage site region, 3-5 amino acids within theinactivation cleavage site region, or 4-5 amino acids within theinactivation cleavage site region.

In aspects of this embodiment, an inactivation cleavage site region ismodified to include an inactivation cleavage site by deleting, adding,substituting, or any combination thereof, e.g., at least 1, 2, 3, 4, or5 amino acids within the inactivation cleavage site region. In otheraspects of this embodiment, an inactivation cleavage site region ismodified to include an inactivation cleavage site by deleting, adding,substituting, or any combination thereof, e.g., at most 1, 2, 3, 4, or 5amino acids within the inactivation cleavage site region. In yet aspectsof this embodiment, an inactivation cleavage site region is modified toinclude an inactivation cleavage site by deleting, adding, substituting,or any combination thereof, e.g., 1-5 amino acids within theinactivation cleavage site region, 1-4 amino acids within theinactivation cleavage site region, 1-3 amino acids within theinactivation cleavage site region, 1-2 amino acids within theinactivation cleavage site region, 2-5 amino acids within theinactivation cleavage site region, 2-4 amino acids within theinactivation cleavage site region, 2-3 amino acids within theinactivation cleavage site region, 3-5 amino acids within theinactivation cleavage site region, or 4-5 amino acids within theinactivation cleavage site region.

Modification of an inactivation cleavage site region to include aninactivation cleavage site can be achieved using standard mutagenesisprocedures known to a person skilled in the art. Non-limiting examplesof mutagenesis procedures, as well as well-characterized reagents,conditions and protocols are readily available from commercial vendorsthat include, without limitation, BD Biosciences-Clontech, Palo Alto,Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc,Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, LaJolla, Calif. These protocols are routine procedures within the scope ofone skilled in the art and from the teaching herein.

As mentioned above, Clostridial toxins and Clostridial toxin chimerasdisclosed in the present specification are translated as single-chainpolypeptides that are subsequently cleaved by proteolytic scissionwithin a disulfide loop region. This posttranslational processing yieldsa di-chain molecule held together by a single disulphide bond andnoncovalent interactions. The proteolytic scission within a disulfideloop region can be achieved by using the endogenous protease cleavagesites naturally-occurring within the di-chain loop region, or byengineering the di-chain loop region to comprise an exogenous proteasecleavage site.

Aspects of the present specification disclose, in part, a di-chain loopregion. As used herein, the term “di-chain loop region” refers to anamino acid sequence of a Clostridial toxin or Clostridial toxin chimericflanked by cysteine amino acids and containing a protease cleavage siteused to convert the single-chain form of a Clostridial toxin orClostridial toxin chimeric into its di-chain form (Table 6).Non-limiting examples of a di-chain loop region, include, a di-chainloop region of BoNT/A comprising amino acids 430-454 of SEQ ID NO: 1; adi-chain loop region of BoNT/B comprising amino acids 437-446 of SEQ IDNO: 2; a di-chain loop region of BoNT/C1 comprising amino acids 437-453of SEQ ID NO: 3; a di-chain loop region of BoNT/D comprising amino acids437-450 of SEQ ID NO: 4; a di-chain loop region of BoNT/E comprisingamino acids 412-426 of SEQ ID NO: 5; a di-chain loop region of BoNT/Fcomprising amino acids 429-445 of SEQ ID NO: 6; a di-chain loop regionof BoNT/G comprising amino acids 436-450 of SEQ ID NO: 7; and a di-chainloop region of TeNT comprising amino acids 439-467 of SEQ ID NO: 8(Table 6).

TABLE 6 Di-chain Loop Region ToxinDi-chain Loop Region Containing the Naturally-occurring Protease Cleavage SiteBoNT/A CVRGIITSKTKSLDKGYNK*----ALNDLC BoNT/BCKSVK*-------------------APGIC BoNT/C1 CHKAIDGRSLYNK*------------TLDCBoNT/D CLRLTKNSR*---------------DDSTC BoNT/ECKNIVSVKGIR*--------------KSIC BoNT/F CKSVIPRKGTK*------------APPRLCBoNT/G CKPVMYKNTGK*--------------SEQC TeNTCKKIIPPTNIRENLYNRTA*SLTDLGGELC BaNT CKS-IVSKKGTK*------------NSLC BuNTCKN-IVSVKGIR*--------------KSIC The amino acid sequence displayed are asfollows: BoNT/A, residues 430-454 of SEQ ID NO: 1; BoNT/B, residues437-446 of SEQ ID NO: 2; BoNT/C1, residues 437-453 of SEQ ID NO: 3;BoNT/D, residues 437-450 of SEQ ID NO: 4; BoNT/E, residues 412-426 ofSEQ ID NO: 5; BoNT/F, residues 429- 445 of SEQ ID NO: 6; BoNT/G,residues 436-450 of SEQ ID NO: 7; TeNT, residues 439-467 of SEQ ID NO:8; BaNT, residues 421-435 of SEQ ID NO: 9; and BuNT, residues 412-426 ofSEQ ID NO: 10. An asterisks (*) indicates the peptide bond that iscleaved by a Clostridial toxin protease.

Thus, in an embodiment, a di-chain loop region comprises a Clostridialtoxin di-chain loop region. In aspects of this embodiment, a di-chainloop region comprises, e.g., a BoNT/A di-chain loop region, a BoNT/Bdi-chain loop region, a BoNT/C1 di-chain loop region, a BoNT/D di-chainloop region, a BoNT/E di-chain loop region, a BoNT/F di-chain loopregion, a BoNT/G di-chain loop region, a TeNT di-chain loop region, aBaNT di-chain loop region, or a BuNT di-chain loop region. In otheraspects of this embodiment, a di-chain loop region comprises, e.g., aBoNT/A di-chain loop region comprising amino acids 430-454 of SEQ ID NO:1; a BoNT/B di-chain loop region comprising amino acids 437-446 of SEQID NO: 2; a BoNT/C1 di-chain loop region comprising amino acids 437-453of SEQ ID NO: 3; a BoNT/D di-chain loop region comprising amino acids437-450 of SEQ ID NO: 4; a BoNT/E di-chain loop region comprising aminoacids 412-426 of SEQ ID NO: 5; a BoNT/F di-chain loop region comprisingamino acids 429-445 of SEQ ID NO: 6; a BoNT/G di-chain loop regioncomprising amino acids 436-450 of SEQ ID NO: 7; or a TeNT di-chain loopregion comprising amino acids 439-467 of SEQ ID NO: 8. a BaNT di-chainloop region comprising amino acids 421-435 of SEQ ID NO: 9; or a BuNTdi-chain loop region comprising amino acids 412-426 of SEQ ID NO: 10.

Aspects of the present specification disclose, in part, an endogenousdi-chain loop protease cleavage site. As used herein, the term“endogenous di-chain loop protease cleavage site” is synonymous with a“naturally occurring di-chain loop protease cleavage site” and refers toa naturally occurring protease cleavage site found within the di-chainloop region of a naturally occurring Clostridial toxin or Clostridialtoxin chimeric and includes, without limitation, naturally occurringClostridial toxin di-chain loop protease cleavage site variants, suchas, e.g., Clostridial toxin di-chain loop protease cleavage siteisoforms and Clostridial toxin di-chain loop protease cleavage sitesubtypes. Non-limiting examples of an endogenous protease cleavage site,include, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/Bdi-chain loop protease cleavage site, a BoNT/C1 di-chain loop proteasecleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/Edi-chain loop protease cleavage site, a BoNT/F di-chain loop proteasecleavage site, a BoNT/G di-chain loop protease cleavage site and a TeNTdi-chain loop protease cleavage site.

While the identity of the protease is currently unknown, the di-chainloop protease cleavage site for many Clostridial toxins has beendetermined. In BoNTs, cleavage at K448-A449 converts the singlepolypeptide form of BoNT/A into the di-chain form; cleavage at K441-A442converts the single polypeptide form of BoNT/B into the di-chain form;cleavage at K449-T450 converts the single polypeptide form of BoNT/C1into the di-chain form; cleavage at R445-D446 converts the singlepolypeptide form of BoNT/D into the di-chain form; cleavage at R422-K423converts the single polypeptide form of BoNT/E into the di-chain form;cleavage at K439-A440 converts the single polypeptide form of BoNT/Finto the di-chain form; and cleavage at K446-5447 converts the singlepolypeptide form of BoNT/G into the di-chain form. Proteolytic cleavageof the single polypeptide form of TeNT at A457-5458 results in thedi-chain form. Proteolytic cleavage of the single polypeptide form ofBaNT at K431-N432 results in the di-chain form. Proteolytic cleavage ofthe single polypeptide form of BuNT at R422—K423 results in the di-chainform. Such a di-chain loop protease cleavage site is operably-linked toa Clostridial toxin or Clostridial toxin chimeric as a fusion protein.However, it should also be noted that additional cleavage sites withinthe di-chain loop also appear to be cleaved resulting in the generationof a small peptide fragment being lost. As a non-limiting example,BoNT/A single-chain polypeptide cleave ultimately results in the loss ofa ten amino acid fragment within the di-chain loop.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric disclosed in the present specification comprises a di-chainloop region including an endogenous di-chain loop protease cleavagesite. In aspects of this embodiment, an endogenous di-chain loopprotease cleavage site located within the di-chain loop regioncomprises, e.g., a BoNT/A di-chain loop protease cleavage site, a BoNT/Bdi-chain loop protease cleavage site, a BoNT/C1 di-chain loop proteasecleavage site, a BoNT/D di-chain loop protease cleavage site, a BoNT/Edi-chain loop protease cleavage site, a BoNT/F di-chain loop proteasecleavage site, a BoNT/G di-chain loop protease cleavage site, a TeNTdi-chain loop protease cleavage site, a BaNT di-chain loop proteasecleavage site, or a BuNT di-chain loop protease cleavage site. In otheraspects of this embodiment, an endogenous di-chain loop proteasecleavage site located within the di-chain loop region comprises, e.g., adi-chain loop region of BoNT/A comprising amino acids 430-454 of SEQ IDNO: 1; a di-chain loop region of BoNT/B comprising amino acids 437-446of SEQ ID NO: 2; a di-chain loop region of BoNT/C1 comprising aminoacids 437-453 of SEQ ID NO: 3; a di-chain loop region of BoNT/Dcomprising amino acids 437-450 of SEQ ID NO: 4; a di-chain loop regionof BoNT/E comprising amino acids 412-426 of SEQ ID NO: 5; a di-chainloop region of BoNT/F comprising amino acids 429-445 of SEQ ID NO: 6; adi-chain loop region of BoNT/G comprising amino acids 436-450 of SEQ IDNO: 7; or a di-chain loop region of TeNT comprising amino acids 439-467of SEQ ID NO: 8. a di-chain loop region of BaNT comprising amino acids421-435 of SEQ ID NO: 9; or a di-chain loop region of BuNT comprisingamino acids 412-426 of SEQ ID NO: 10.

Aspects of the present specification disclose, in part, an exogenousprotease cleavage site. As used herein, the term “exogenous proteasecleavage site” is synonymous with “engineered protease cleavage site”,“non-naturally occurring protease cleavage site”, or “non-nativeprotease cleavage site” and refers to a protease cleavage site that isnot normally present in a di-chain loop region from a naturallyoccurring Clostridial toxin. Such engineered or exogenous proteasecleavage sites within the di-chain loop region are used to convert thesingle-chain polypeptide form of a Clostridial toxin of Clostridialtoxin chimeric disclosed in the present specification into its di-chainform. It is envisioned that any and all exogenous protease cleavagesites can be used to convert the single-chain polypeptide form of aClostridial toxin or Clostridial toxin chimeric into its active di-chainform are useful to practice aspects of the present specification.Non-limiting examples of exogenous protease cleavage sites include,e.g., a plant papain cleavage site, an insect papain cleavage site, acrustacean papain cleavage site, an enterokinase cleavage site, a humanrhinovirus 3C protease cleavage site, a human enterovirus 3C proteasecleavage site, a tobacco etch virus (TEV) protease cleavage site, aTobacco Vein Mottling Virus (TVMV) cleavage site, a subtilisin cleavagesite, a hydroxylamine cleavage site, or a Caspase 3 cleavage site.Engineered protease cleavage sites located within the di-chain loop aredescribed in, e.g., Dolly, et al., Activatable Recombinant Neurotoxins,U.S. Pat. No. 7,419,676, Dolly, et al., Activatable RecombinantNeurotoxins, U.S. Pat. No. 7,422,877, Steward, et al., ActivatableRecombinant Neurotoxins, U.S. Patent Publication 2009/0069238, Steward,et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication2008/0032930, Steward, et al., Activatable Recombinant Neurotoxins, U.S.Patent Publication 2009/0018081, Steward, et al., ActivatableRecombinant Neurotoxins, U.S. Patent Publication 2009/0005313, Steward,et al., Activatable Recombinant Neurotoxins, U.S. Patent Publication2009/0004224; each of which is hereby incorporated by reference in itsentirety.

It is envisioned that an exogenous protease cleavage site of any and alllengths can be useful in aspects of the present specification with theproviso that the exogenous protease cleavage site can be cleaved by itsrespective protease. Thus, in aspects of this embodiment, an exogenousprotease cleavage site can have a length of, e.g., at least 6, 7, 8, 9,10, 15, 20, 25, 30, 40, 50, or at least 60 amino acids; or at most 6, 7,8, 9, 10, 15, 20, 25, 30, 40, 50, or at least 60 amino acids.

In an embodiment, a Clostridial toxin or Clostridial toxin chimericdisclosed in the present specification comprises a di-chain loop regionincluding an exogenous protease cleavage site. In aspects of thisembodiment, an exogenous protease cleavage site located within thedi-chain loop region comprises, e.g., a plant papain cleavage site, aninsect papain cleavage site, a crustacean papain cleavage site, anon-human enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Tobacco Vein Mottling Virus protease cleavagesite, a human rhinovirus 3C protease cleavage site, a human enterovirus3C protease cleavage site, a subtilisin cleavage site, a hydroxylaminecleavage site, a SUMO/ULP-1 protease cleavage site, and a non-humanCaspase 3 cleavage site.

In an aspect of this embodiment, an exogenous protease cleavage sitelocated within the di-chain loop region comprises, e.g., a non-humanenterokinase cleavage site. In another aspect of the embodiment, anexogenous protease cleavage site located within the di-chain loop regioncomprises, e.g., a bovine enterokinase protease cleavage site. In yetanother aspect of the embodiment, an exogenous protease cleavage sitelocated within the di-chain loop region comprises, e.g., SEQ ID NO: 480.

In another aspect of this embodiment, an exogenous protease cleavagesite located within the di-chain loop region comprises, e.g., a TobaccoEtch Virus protease cleavage. In other aspects of the embodiment, anexogenous protease cleavage site located within the di-chain loop regioncomprises, e.g., the consensus sequence EX₁X₂YX₃Q*G (SEQ ID NO: 481) orEX₁X₂YX₃Q*S (SEQ ID NO: 482), where X₁, X₂ and X₃ is any amino acid. Inother aspects of the embodiment, an exogenous protease cleavage sitelocated within the di-chain loop region comprises, e.g., SEQ ID NO: 483,SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ IDNO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, or SEQ ID NO:492.

In another aspect of this embodiment, an exogenous protease cleavagesite located within the di-chain loop region comprises, e.g., a TobaccoVein Mottling Virus protease cleavage site. In other aspects of theembodiment, an exogenous protease cleavage site located within thedi-chain loop region comprises, e.g., the consensus sequence X₁X₂VRFQ*G(SEQ ID NO: 493) or X₁X₂VRFQ*S (SEQ ID NO: 494), where X₁ and X₂ areindependently any amino acid. In other aspects of the embodiment, anexogenous protease cleavage site located within the di-chain loop regioncomprises, e.g., SEQ ID NO: 495, SEQ ID NO: 496, SEQ ID NO: 497, or SEQID NO: 498.

In still another aspect of this embodiment, an exogenous proteasecleavage site located within the di-chain loop region comprises, e.g., ahuman rhinovirus 3C protease cleavage site. In another aspect of theembodiment, an exogenous protease cleavage site located within thedi-chain loop region comprises, e.g., the consensus sequence X₁X₂LFQ*GP(SEQ ID NO: 499), where X₁ is any amino acid with an acidic amino acidlike D or E preferred; and X₂ is preferentially S, T, and an aliphatichydrophobic amino acid like G, P, A, V, L, I, and M. In other aspects ofthe embodiment, an exogenous protease cleavage site located within thedi-chain loop region comprises, e.g., SEQ ID NO: 500, SEQ ID NO: 501,SEQ ID NO: 502, SEQ ID NO: 503, SEQ ID NO: 504, or SEQ ID NO: 505. Inanother aspect of the embodiment, an exogenous protease cleavage sitelocated within the di-chain loop region comprises, e.g., a humanrhinovirus 3C protease cleaved by PRESCISSION®.

In yet another aspect of this embodiment, an exogenous protease cleavagesite located within the di-chain loop region comprises, e.g., asubtilisin cleavage site. In other aspects of the embodiment, anexogenous protease cleavage site located within the di-chain loop regioncomprises, e.g., the consensus sequence X₁X₂X₃X₄WY (SEQ ID NO: 506) orX₁X₂X₃X₄YH* (SEQ ID NO: 507), where X₁, X₂, X₃, and X₄ are independentlyany amino acid. In other aspects of the embodiment, an exogenousprotease cleavage site located within the di-chain loop regioncomprises, e.g., SEQ ID NO: 508, SEQ ID NO: 509, or SEQ ID NO: 510. Inother aspects of the embodiment, an exogenous protease cleavage sitelocated within the di-chain loop region comprises, e.g., a subtilisincleavage site cleaved by GENENASE®.

In yet another aspect of this embodiment, an exogenous protease cleavagesite located within the di-chain loop region comprises, e.g., ahydroxylamine cleavage site. In other aspects of the embodiment, anexogenous protease cleavage site located within the di-chain loop regioncomprises, e.g., the dipeptide N*G. In other aspects of the embodiment,an exogenous protease cleavage site located within the di-chain loopregion comprises, e.g., SEQ ID NO: 511 or SEQ ID NO: 512.

In yet another aspect of this embodiment, an exogenous protease cleavagesite located within the di-chain loop region comprises, e.g., aSUMO/ULP-1 protease cleavage site. In other aspects of the embodiment,an exogenous protease cleavage site located within the di-chain loopregion comprises, e.g., the consensus sequence GG*X₁X₂X₃ (SEQ ID NO:513), where X₁, X₂, and X₃ are independently any amino acid. In otheraspects of the embodiment, an exogenous protease cleavage site locatedwithin the di-chain loop region comprises, e.g., SEQ ID NO: 514.

In an aspect of this embodiment, an exogenous protease cleavage sitelocated within the di-chain loop region comprises, e.g., a Caspase 3cleavage site. In other aspects of the embodiment, an exogenous proteasecleavage site located within the di-chain loop region comprises, e.g., anon-human Caspase 3 protease cleavage site. In other aspects of theembodiment, an exogenous protease cleavage site located within thedi-chain loop region comprises, e.g., the consensus sequence DX₁X₂D*X₃(SEQ ID NO: 515), where X₁ is any amino acid, with an acidic amino acidlike D and E preferred, X₂ is any amino acid and X₃ is amino acid, witha small non-polar amino acid like A, C, G, S, and T preferred. In otheraspects of the embodiment, an exogenous protease cleavage site locatedwithin the di-chain loop region comprises, e.g., SEQ ID NO: 516, SEQ IDNO: 517, SEQ ID NO: 518, SEQ ID NO: 519, SEQ ID NO: 520, or SEQ ID NO:521.

A di-chain loop region can be modified so that a naturally-occurringdi-chain loop protease cleavage site is replaced by an exogenousprotease cleavage site. In this modification, the naturally-occurringdi-chain loop protease cleavage site is made inoperable and thus cannotbe cleaved by its protease. Only the exogenous protease cleavage sitecan be cleaved by its corresponding exogenous protease. In this type ofmodification, the exogenous protease site is operably-linked to aClostridial toxin or Clostridial toxin chimeric as a fusion protein andthe site can be cleaved by its respective exogenous protease.Replacement of an endogenous di-chain loop protease cleavage site withan exogenous protease cleavage site can be a substitution of the siteswhere the exogenous site is engineered at the position approximating thecleavage site location of the endogenous site. Replacement of anendogenous di-chain loop protease cleavage site with an exogenousprotease cleavage site can be an addition of an exogenous site where theexogenous site is engineered at the position different from the cleavagesite location of the endogenous site, the endogenous site beingengineered to be inoperable. The location and kind of protease cleavagesite may be critical because certain binding domains require a freeamino-terminal or carboxyl-terminal amino acid. For example, when apeptide binding domain is placed between two other domains, e.g., seeFIG. 4, a criterion for selection of a protease cleavage site could bewhether the protease that cleaves its site leaves a flush cut, exposingthe free amino-terminal or carboxyl-terminal of the binding domainnecessary for selective binding of the binding domain to its receptor.

A naturally-occurring protease cleavage site can be made inoperable byaltering at least one of the two amino acids flanking the peptide bondcleaved by the naturally-occurring di-chain loop protease. Moreextensive alterations can be made, with the proviso that the twocysteine residues of the di-chain loop region remain intact and theregion can still form the disulfide bridge. Non-limiting examples of anamino acid alteration include deletion of an amino acid or replacementof the original amino acid with a different amino acid. Thus, in oneembodiment, a naturally-occurring protease cleavage site is madeinoperable by altering at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20amino acids including at least one of the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease. In anotherembodiment, a naturally-occurring protease cleavage site is madeinoperable by altering at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20amino acids including at least one of the two amino acids flanking thepeptide bond cleaved by a naturally-occurring protease.

It is understood that a modified Clostridial toxin disclosed in thepresent specification can optionally further comprise a flexible regioncomprising a flexible spacer. A flexible region comprising flexiblespacers can be used to adjust the length of a polypeptide region inorder to optimize a characteristic, attribute or property of apolypeptide. As a non-limiting example, a polypeptide region comprisingone or more flexible spacers in tandem can be used to better expose aprotease cleavage site thereby facilitating cleavage of that site by aprotease. As another non-limiting example, a polypeptide regioncomprising one or more flexible spacers in tandem can be used to betterpresent a peptide binding domain, thereby facilitating the binding ofthat binding domain to its receptor.

A flexible space comprising a peptide is at least one amino acid inlength and comprises non-charged amino acids with small side-chain Rgroups, such as, e.g., small non-polar amino acids like A, C, G, S, andT. Thus, in an embodiment a flexible spacer can have a length of, e.g.,at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; or at most 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In still another embodiment, aflexible spacer can be, e.g., between 1-3 amino acids, between 2-4 aminoacids, between 3-5 amino acids, between 4-6 amino acids, or between 5-7amino acids. Non-limiting examples of a flexible spacer include, e.g., aG-spacers such as GGG, GGGG (SEQ ID NO: 522), and GGGGS (SEQ ID NO: 523)or an A-spacers such as AAA, AAAA (SEQ ID NO: 524) and AAAAT (SEQ ID NO:525). Such a flexible region is operably-linked in-frame to the modifiedClostridial toxin as a fusion protein.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric disclosed in the present specification can further comprise aflexible region comprising a flexible spacer. In another embodiment, aClostridial toxin or Clostridial toxin chimeric disclosed in the presentspecification can further comprise flexible region comprising aplurality of flexible spacers in tandem. In aspects of this embodiment,a flexible region can comprise in tandem, e.g., at least 1, 2, 3, 4, or5 G-spacers; or at most 1, 2, 3, 4, or 5 G-spacers. In still otheraspects of this embodiment, a flexible region can comprise in tandem,e.g., at least 1, 2, 3, 4, or 5 A-spacers; or at most 1, 2, 3, 4, or 5A-spacers. In another aspect of this embodiment, a Clostridial toxin orClostridial toxin chimeric can comprise a flexible region comprising oneor more copies of the same flexible spacers, one or more copies ofdifferent flexible-spacer regions, or any combination thereof.

It is envisioned that a Clostridial toxin or Clostridial toxin chimericdisclosed in the present specification can comprise a flexible spacer inany and all locations with the proviso that the Clostridial toxin orClostridial toxin chimeric is capable of performing the overallintoxication process. In aspects of this embodiment, a flexible spaceris positioned between, e.g., an enzymatic domain and a translocationdomain, an enzymatic domain and a binding domain, an enzymatic domainand an exogenous protease cleavage site. In other aspects of thisembodiment, a flexible spacer is positioned between, e.g., a bindingdomain and a translocation domain, a binding domain and an enzymaticdomain, a binding domain and an exogenous protease cleavage site. In yetother aspects of this embodiment, a flexible spacer is positionedbetween, e.g., a translocation domain and an enzymatic domain, atranslocation domain and a binding domain, a translocation domain and anexogenous protease cleavage site.

As another non-limiting example of an optional component, a Clostridialtoxin or Clostridial toxin chimeric can further comprise anepitope-binding region. An epitope-binding region can be used in a widevariety of procedures involving, e.g., protein purification and proteinvisualization. Such an epitope-binding region is operably-linkedin-frame to a modified Clostridial toxin as a fusion protein.Non-limiting examples of an epitope-binding region include, e.g., FLAG,Express™, human Influenza virus hemagglutinin (HA), human p62^(c-Myc)protein (c-MYC), Vesicular Stomatitis Virus Glycoprotein (VSV-G),glycoprotein-D precursor of Herpes simplex virus (HSV), V5, AU1, andAU5; affinity-binding, such as. e.g., polyhistidine (HIS), streptavidinbinding peptide (strep), and biotin or a biotinylation sequence;peptide-binding regions, such as. e.g., the glutathione binding domainof glutathione-S-transferase, the calmodulin binding domain of thecalmodulin binding protein, and the maltose binding domain of themaltose binding protein. Non-limiting examples of specific protocols forselecting, making and using an appropriate binding peptide are describedin, e.g., Epitope Tagging, pp. 17.90-17.93 (Sambrook and Russell, eds.,MOLECULAR CLONING A LABORATORY MANUAL, Vol. 3, 3^(rd) ed. 2001);ANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane, eds., ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1998); and USING ANTIBODIES:A LABORATORY MANUAL: PORTABLE PROTOCOL NO. I (Edward Harlow & DavidLane, Cold Spring Harbor Laboratory Press, 1998). In addition,non-limiting examples of binding peptides as well as well-characterizedreagents, conditions and protocols are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene,La Jolla, Calif. These protocols are routine procedures well within thescope of one skilled in the art and from the teaching herein.

Thus, in an embodiment, a Clostridial toxin or Clostridial toxinchimeric disclosed in the present specification can further comprise anepitope-binding region. In another embodiment, a Clostridial toxin orClostridial toxin chimeric disclosed in the present specification canfurther comprises a plurality of epitope-binding regions. In aspects ofthis embodiment, a Clostridial toxin or Clostridial toxin chimeric cancomprise, e.g., at least 1, 2, 3, 4, or 5 epitope-binding regions. Inother aspects of this embodiment, a Clostridial toxin or Clostridialtoxin chimeric can comprise, e.g., at most 1, 2, 3, 4, or 5epitope-binding regions. In another aspect of this embodiment, amodified Clostridial toxin can comprise one or more copies of the sameepitope-binding region, one or more copies of different epitope-bindingregions, or any combination thereof.

The location of an epitope-binding region can be in various positions,including, without limitation, at the amino terminus, within, or at thecarboxyl terminus of a Clostridial toxin or Clostridial toxin chimeric.Thus, in an embodiment, an epitope-binding region is located at theamino-terminus of a Clostridial toxin or Clostridial toxin chimeric. Inanother embodiment, an epitope-binding region is located at thecarboxyl-terminus of a modified Clostridial toxin.

Aspects of the present specification provide, in part, polynucleotidemolecules. As used herein, the term “polynucleotide molecule” issynonymous with “nucleic acid molecule” and refers to a polymeric formof nucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides.It is envisioned that any and all polynucleotide molecules that canencode a Clostridial toxin or Clostridial toxin chimeric disclosed inthe present specification can be useful, including, without limitationnaturally-occurring and non-naturally-occurring DNA molecules andnaturally-occurring and non-naturally-occurring RNA molecules.Non-limiting examples of naturally-occurring and non-naturally-occurringDNA molecules include single-stranded DNA molecules, double-stranded DNAmolecules, genomic DNA molecules, cDNA molecules, vector constructs,such as, e.g., plasmid constructs, phagemid constructs, bacteriophageconstructs, retroviral constructs and artificial chromosome constructs.Non-limiting examples of naturally-occurring and non-naturally-occurringRNA molecules include single-stranded RNA, double stranded RNA and mRNA.

Well-established molecular biology techniques that may be necessary tomake a polynucleotide molecule encoding a Clostridial toxin orClostridial toxin chimeric disclosed in the present specificationincluding, but not limited to, procedures involving polymerase chainreaction (PCR) amplification, restriction enzyme reactions, agarose gelelectrophoresis, nucleic acid ligation, bacterial transformation,nucleic acid purification, nucleic acid sequencing andrecombination-based techniques are routine procedures well within thescope of one skilled in the art and from the teaching herein.Non-limiting examples of specific protocols necessary to make apolynucleotide molecule encoding a modified Clostridial toxin aredescribed in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al.,eds. John Wiley & Sons, 2004). Additionally, a variety of commerciallyavailable products useful for making a polynucleotide molecule encodinga Clostridial toxin or Clostridial toxin chimeric disclosed in thepresent specification are widely available. These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

Thus, in an embodiment, a polynucleotide molecule encodes a Clostridialtoxin or Clostridial toxin chimeric disclosed in the presentspecification.

Another aspect of the present specification provides, in part, a methodof producing a Clostridial toxin or Clostridial toxin chimeric disclosedin the present specification, such method comprising the step ofexpressing a polynucleotide molecule encoding a Clostridial toxin orClostridial toxin chimeric in a cell. Another aspect of the presentspecification provides a method of producing a Clostridial toxin orClostridial toxin chimeric disclosed in the present specification, suchmethod comprising the steps of introducing an expression constructcomprising a polynucleotide molecule encoding a Clostridial toxin orClostridial toxin chimeric into a cell and expressing the expressionconstruct in the cell.

The methods disclosed in the present specification include, in part, aClostridial toxin or Clostridial toxin chimeric. It is envisioned thatany and all Clostridial toxins or Clostridial toxin chimeras disclosedin the present specification can be produced using the methods disclosedin the present specification. It is also envisioned that any and allpolynucleotide molecules encoding a Clostridial toxin or Clostridialtoxin chimeric disclosed in the present specification can be useful inproducing a Clostridial toxin or Clostridial toxin chimeric disclosed inthe present specification using the methods disclosed in the presentspecification.

The methods disclosed in the present specification include, in part, anexpression construct. An expression construct comprises a polynucleotidemolecule disclosed in the present specification operably-linked to anexpression vector useful for expressing the polynucleotide molecule in acell or cell-free extract. A wide variety of expression vectors can beemployed for expressing a polynucleotide molecule encoding a Clostridialtoxin or Clostridial toxin chimeric disclosed in the presentspecification, including, without limitation, a viral expression vector;a prokaryotic expression vector; eukaryotic expression vectors, such as,e.g., a yeast expression vector, an insect expression vector and amammalian expression vector; and a cell-free extract expression vector.It is further understood that expression vectors useful to practiceaspects of these methods may include those which express a Clostridialtoxin or Clostridial toxin chimeric under control of a constitutive,tissue-specific, cell-specific or inducible promoter element, enhancerelement or both. Non-limiting examples of expression vectors, along withwell-established reagents and conditions for making and using anexpression construct from such expression vectors are readily availablefrom commercial vendors that include, without limitation, BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMDBiosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; andStratagene, La Jolla, Calif. The selection, making and use of anappropriate expression vector are routine procedures well within thescope of one skilled in the art and from the teachings herein.

Thus, in aspects of this embodiment, a polynucleotide molecule encodinga Clostridial toxin or Clostridial toxin chimeric disclosed in thepresent specification operably-linked to an expression vector. Inaspects of this embodiment, the expression vector is, e.g., a viralexpression vector, a prokaryotic expression vector, a yeast expressionvector, an insect expression vector, or a mammalian expression vector.On other aspects of this embodiment, a polynucleotide molecule encodinga Clostridial toxin or Clostridial toxin chimeric disclosed in thepresent specification operably-linked to a cell-free extract expressionvector.

The methods disclosed in the present specification include, in part, acell. It is envisioned that any and all cells can be used. Thus, aspectsof this embodiment include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacteria cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection, European Collection of Cell Cultures and the GermanCollection of Microorganisms and Cell Cultures. Non-limiting examples ofspecific protocols for selecting, making and using an appropriate cellline are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer AcademicPublishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUESOF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUECULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley andSons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press,2^(nd) ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra,(2004). These protocols are routine procedures within the scope of oneskilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part,introducing into a cell a polynucleotide molecule. A polynucleotidemolecule introduced into a cell can be transiently or stably maintainedby that cell. Stably-maintained polynucleotide molecules may beextra-chromosomal and replicate autonomously, or they may be integratedinto the chromosomal material of the cell and replicatenon-autonomously. It is envisioned that any and all methods forintroducing a polynucleotide molecule disclosed in the presentspecification into a cell can be used. Methods useful for introducing apolynucleotide molecule into a cell include, without limitation,chemical-mediated transfection or transformation such as, e.g., calciumchloride-mediated, calcium phosphate-mediated, diethyl-aminoethyl (DEAE)dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated,polylysine-mediated and polybrene-mediated; physical-mediatedtransfection or transformation, such as, e.g., biolistic particledelivery, microinjection, protoplast fusion and electroporation; andviral-mediated transfection, such as, e.g., retroviral-mediatedtransfection, see, e.g., Introducing Cloned Genes into CulturedMammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., MolecularCloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). One skilled inthe art understands that selection of a specific method to introduce anexpression construct into a cell will depend, in part, on whether thecell will transiently contain an expression construct or whether thecell will stably contain an expression construct. These protocols areroutine procedures within the scope of one skilled in the art and fromthe teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termedtransfection, is used to introduce into a cell a polynucleotide moleculeencoding a Clostridial toxin or Clostridial toxin chimeric disclosed inthe present specification. In chemical-mediated methods of transfectionthe chemical reagent forms a complex with the nucleic acid thatfacilitates its uptake into the cells. Such chemical reagents include,without limitation, calcium phosphate-mediated, see, e.g., Martin Jordan& Florian Worm, Transfection of adherent and suspended cells by calciumphosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE)dextran-mediated, lipid-mediated, cationic polymer-mediated likepolyethyleneimine (PEI)-mediated and polylysine-mediated andpolybrene-mediated, see, e.g., Chun Zhang et al., Polyethyleniminestrategies for plasmid delivery to brain-derived cells, 33(2) Methods144-150 (2004). Such chemical-mediated delivery systems can be preparedby standard methods and are commercially available, see, e.g., CellPhectTransfection Kit (Amersham Biosciences, Piscataway, N.J.); MammalianTransfection Kit, Calcium phosphate and DEAE Dextran, (Stratagene, Inc.,La Jolla, Calif.); LIPOFECTAMINE™ Transfection Reagent (Invitrogen,Inc., Carlsbad, Calif.); ExGen 500 Transfection kit (Fermentas, Inc.,Hanover, Md.), and SuperFect and Effectene Transfection Kits (Qiagen,Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is usedto introduce into a cell a polynucleotide molecule encoding aClostridial toxin or Clostridial toxin chimeric disclosed in the presentspecification. Physical techniques include, without limitation,electroporation, biolistic and microinjection. Biolistics andmicroinjection techniques perforate the cell wall in order to introducethe nucleic acid molecule into the cell, see, e.g., Jeike E. Biewenga etal., Plasmid-mediated gene transfer in neurons using the biolisticstechnique, 71(1) J. Neurosci. Methods 67-75 (1997); and John O'Brien &Sarah C. R. Lummis, Biolistic and diolistic transfection: using the genegun to deliver DNA and lipophilic dyes into mammalian cells, 33(2)Methods 121-125 (2004). Electroporation, also termedelectropermeabilization, uses brief, high-voltage, electrical pulses tocreate transient pores in the membrane through which the nucleic acidmolecules enter and can be used effectively for stable and transienttransfections of all cell types, see, e.g., M. Golzio et al., In vitroand in vivo electric field-mediated permeabilization, gene transfer, andexpression, 33(2) Methods 126-135 (2004); and Oliver Gresch et al., Newnon-viral method for gene transfer into primary cells, 33(2) Methods151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termedtransduction, is used to introduce into a cell a polynucleotide moleculeencoding a Clostridial toxin or Clostridial toxin chimeric disclosed inthe present specification. In viral-mediated methods of transienttransduction, the process by which viral particles infect and replicatein a host cell has been manipulated in order to use this mechanism tointroduce a polynucleotide molecule into the cell. Viral-mediatedmethods have been developed from a wide variety of viruses including,without limitation, retroviruses, adenoviruses, adeno-associatedviruses, herpes simplex viruses, picornaviruses, alphaviruses andbaculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV basedretroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods164-172 (2004); and Maurizio Federico, From lentiviruses to lentivirusvectors, 229 Methods Mol. Biol. 3-15 (2003); E. M. Poeschla, Non-primatelentiviral vectors, 5(5) Curr. Opin. Mol. Ther. 529-540 (2003); KarimBenihoud et al, Adenovirus vectors for gene delivery, 10(5) Curr. Opin.Biotechnol. 440-447 (1999); H. Bueler, Adeno-associated viral vectorsfor gene transfer and gene therapy, 380(6) Biol. Chem. 613-622 (1999);Chooi M. Lai et al., Adenovirus and adeno-associated virus vectors,21(12) DNA Cell Biol. 895-913 (2002); Edward A. Burton et al., Genedelivery using herpes simplex virus vectors, 21(12) DNA Cell Biol.915-936 (2002); Paola Grandi et al., Targeting HSV amplicon vectors,33(2) Methods 179-186 (2004); Ilya Frolov et al., Alphavirus-basedexpression vectors: strategies and applications, 93(21) Proc. Natl.Acad. Sci. U.S.A. 11371-11377 (1996); Markus U. Ehrengruber, Alphaviralgene transfer in neurobiology, 59(1) Brain Res. Bull. 13-22 (2002);Thomas A. Kost & J. Patrick Condreay, Recombinant baculoviruses asmammalian cell gene-delivery vectors, 20(4) Trends Biotechnol. 173-180(2002); and A. Huser & C. Hofmann, Baculovirus vectors: novel mammaliancell gene-delivery vehicles and their applications, 3(1) Am. J.Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, areoften selected for mammalian cell transduction because adenoviruseshandle relatively large polynucleotide molecules of about 36 kb, areproduced at high titer, and can efficiently infect a wide variety ofboth dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermenset al., Transient gene transfer to neurons and glia: analysis ofadenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci.Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches forgenerating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev.165-176 (2001). Transduction using adenoviral-based system do notsupport prolonged protein expression because the nucleic acid moleculeis carried by an episome in the cell nucleus, rather than beingintegrated into the host cell chromosome. Adenoviral vector systems andspecific protocols for how to use such vectors are disclosed in, e.g.,VIRAPOWER™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and VIRAPOWER™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and ADEASY™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andADEASY™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc.

Polynucleotide molecule delivery can also use single-stranded RNAretroviruses, such as, e.g., oncoretroviruses and lentiviruses.Retroviral-mediated transduction often produce transduction efficienciesclose to 100%, can easily control the proviral copy number by varyingthe multiplicity of infection (MOI), and can be used to eithertransiently or stably transduce cells, see, e.g., Tiziana Tonini et al.,Transient production of retroviral-and lentiviral-based vectors for thetransduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004);Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivoand in vivo gene transfer, 33(2) Methods 164-172 (2004); FélixRecillas-Targa, Gene transfer and expression in mammalian cell lines andtransgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and RolandWolkowicz et al., Lentiviral vectors for the delivery of DNA intomammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviralparticles consist of an RNA genome packaged in a protein capsid,surrounded by a lipid envelope. The retrovirus infects a host cell byinjecting its RNA into the cytoplasm along with the reversetranscriptase enzyme. The RNA template is then reverse transcribed intoa linear, double stranded cDNA that replicates itself by integratinginto the host cell genome. Viral particles are spread both vertically(from parent cell to daughter cells via the provirus) as well ashorizontally (from cell to cell via virions). This replication strategyenables long-term persistent expression since the nucleic acid moleculesof interest are stably integrated into a chromosome of the host cell,thereby enabling long-term expression of the protein. For instance,animal studies have shown that lentiviral vectors injected into avariety of tissues produced sustained protein expression for more than 1year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector, 272(5259)Science 263-267 (1996). The Oncoretroviruses-derived vector systems,such as, e.g., Moloney murine leukemia virus (MoMLV), are widely usedand infect many different non-dividing cells. Lentiviruses can alsoinfect many different cell types, including dividing and non-dividingcells and possess complex envelope proteins, which allows for highlyspecific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectorsare disclosed in, e.g., Manfred Gossen & Hermann Bujard, Tight controlof gene expression in eukaryotic cells by tetracycline-responsivepromoters, U.S. Pat. No. 5,464,758 (Nov. 7, 1995) and Hermann Bujard &Manfred Gossen, Methods for regulating gene expression, U.S. Pat. No.5,814,618 (Sep. 29, 1998) David S. Hogness, Polynucleotides encodinginsect steroid hormone receptor polypeptides and cells transformed withsame, U.S. Pat. No. 5,514,578 (May 7, 1996) and David S. Hogness,Polynucleotide encoding insect ecdysone receptor, U.S. Pat. No.6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al., Progesteronereceptor having C. terminal hormone binding domain truncations, U.S.Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegeto et al., Mutatedsteroid hormone receptors, methods for their use and molecular switchfor gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23, 1999) and ElisabettaVegeto et al., Mutated steroid hormone receptors, methods for their useand molecular switch for gene therapy, U.S. Pat. No. 5,935,934 (Aug. 10,1999). Furthermore, such viral delivery systems can be prepared bystandard methods and are commercially available, see, e.g., BD™ Tet-Offand Tet-On Gene Expression Systems (BD Biosciences-Clontech, Palo Alto,Calif.) and BD™ Tet-Off and Tet-On Gene Expression Systems User Manual,PT3001-1, BD Biosciences Clontech, (Mar. 14, 2003), GENESWITCH™ System(Invitrogen, Inc., Carlsbad, Calif.) and GENESWITCH™ System AMifepristone-Regulated Expression System for Mammalian Cells version D,25-0313, Invitrogen, Inc., (Nov. 4, 2002); VIRAPOWER™ LentiviralExpression System (Invitrogen, Inc., Carlsbad, Calif.) and VIRAPOWER™Lentiviral Expression System Instruction Manual 25-0501 version E,Invitrogen, Inc., (Dec. 8, 2003); and COMPLETE CONTROL® RetroviralInducible Mammalian Expression System (Stratagene, La Jolla, Calif.) andCOMPLETE CONTROL® Retroviral Inducible Mammalian Expression SystemInstruction Manual, 064005e.

The methods disclosed in the present specification include, in part,expressing from a polynucleotide molecule a Clostridial toxin orClostridial toxin chimeric disclosed in the present specification. It isenvisioned that any of a variety of expression systems may be useful forexpressing from a polynucleotide molecule a Clostridial toxin orClostridial toxin chimeric disclosed in the present specification,including, without limitation, cell-based systems and cell-freeexpression systems. Cell-based systems include, without limitation,viral expression systems, prokaryotic expression systems, yeastexpression systems, baculoviral expression systems, insect expressionsystems, and mammalian expression systems. Cell-free systems include,without limitation, wheat germ extracts, rabbit reticulocyte extracts,and E. coli extracts and generally are equivalent to the methoddisclosed herein. Expression of a polynucleotide molecule using anexpression system can include any of a variety of characteristicsincluding, without limitation, inducible expression, non-inducibleexpression, constitutive expression, viral-mediated expression,stably-integrated expression, and transient expression. Expressionsystems that include well-characterized vectors, reagents, conditionsand cells are well-established and are readily available from commercialvendors that include, without limitation, Ambion, Inc. Austin, Tex.; BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; QIAGEN, Inc.,Valencia, Calif.; Roche Applied Science, Indianapolis, Ind.; andStratagene, La Jolla, Calif. Non-limiting examples on the selection anduse of appropriate heterologous expression systems are described ine.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B.David Hames eds., Oxford University Press, 1999); Joseph M. Fernandez &James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OFEXPRESSION (Academic Press, 1999); and Meena Rai & Harish Padh,Expression Systems for Production of Heterologous Proteins, 80(9)CURRENT SCIENCE 1121-1128, (2001). These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

A variety of cell-based expression procedures are useful for expressinga polynucleotide molecule encoding a Clostridial toxin or Clostridialtoxin chimeric disclosed in the present specification. Examplesincluded, without limitation, viral expression systems, prokaryoticexpression systems, yeast expression systems, baculoviral expressionsystems, insect expression systems, and mammalian expression systems.Viral expression systems include, without limitation, the VIRAPOWER™Lentiviral (Invitrogen, Inc., Carlsbad, Calif.), the AdenoviralExpression Systems (Invitrogen, Inc., Carlsbad, Calif.), the ADEASY™ XLAdenoviral Vector System (Stratagene, La Jolla, Calif.) and theVIRAPORT® Retroviral Gene Expression System (Stratagene, La Jolla,Calif.). Non-limiting examples of prokaryotic expression systems includethe CHAMPION™ pET Expression System (EMD Biosciences-Novagen, Madison,Wis.), the TRIEX™ Bacterial Expression System (EMD Biosciences-Novagen,Madison, Wis.), the QIAEXPRESS® Expression System (QIAGEN, Inc.), andthe AFFINITY® Protein Expression and Purification System (Stratagene, LaJolla, Calif.). Yeast expression systems include, without limitation,the EASYSELECT™ Pichia Expression Kit (Invitrogen, Inc., Carlsbad,Calif.), the YES-ECHO™ Expression Vector Kits (Invitrogen, Inc.,Carlsbad, Calif.) and the SPECTRA™ S. pombe Expression System(Invitrogen, Inc., Carlsbad, Calif.). Non-limiting examples ofbaculoviral expression systems include the BACULODIRECT™ (Invitrogen,Inc., Carlsbad, Calif.), the BAC-TO-BAC® (Invitrogen, Inc., Carlsbad,Calif.), and the BD BACULOGOLD™ (BD Biosciences-Pharmingen, San Diego,Calif.). Insect expression systems include, without limitation, theDrosophila Expression System (DES®) (Invitrogen, Inc., Carlsbad,Calif.), INSECTSELECT™ System (Invitrogen, Inc., Carlsbad, Calif.) andINSECTDIRECT™ System (EMD Biosciences-Novagen, Madison, Wis.).Non-limiting examples of mammalian expression systems include the T-REX™(Tetracycline-Regulated Expression) System (Invitrogen, Inc., Carlsbad,Calif.), the FLP-IN™ T-REX™ System (Invitrogen, Inc., Carlsbad, Calif.),the pcDNA™ system (Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2system (Invitrogen, Inc., Carlsbad, Calif.), the EXCHANGER® System,INTERPLAY™ Mammalian TAP System (Stratagene, La Jolla, Calif.), COMPLETECONTROL® Inducible Mammalian Expression System (Stratagene, La Jolla,Calif.) and LACSWITCH® II Inducible Mammalian Expression System(Stratagene, La Jolla, Calif.).

Another procedure of expressing a polynucleotide molecule encoding aClostridial toxin or Clostridial toxin chimeric disclosed in the presentspecification employs a cell-free expression system such as, withoutlimitation, prokaryotic extracts and eukaryotic extracts. Non-limitingexamples of prokaryotic cell extracts include the RTS 100 E. coli HY Kit(Roche Applied Science, Indianapolis, Ind.), the ActivePro In VitroTranslation Kit (Ambion, Inc., Austin, Tex.), the ECOPRO™ System (EMDBiosciences-Novagen, Madison, Wis.) and the EXPRESSWAY™ Plus ExpressionSystem (Invitrogen, Inc., Carlsbad, Calif.). Eukaryotic cell extractinclude, without limitation, the RTS 100 Wheat Germ CECF Kit (RocheApplied Science, Indianapolis, Ind.), the TNT® Coupled Wheat GermExtract Systems (Promega Corp., Madison, Wis.), the Wheat Germ IVT™ Kit(Ambion, Inc., Austin, Tex.), the Retic Lysate IVT™ Kit (Ambion, Inc.,Austin, Tex.), the PROTEINSCRIPT® II System (Ambion, Inc., Austin, Tex.)and the TNT® Coupled Reticulocyte Lysate Systems (Promega Corp.,Madison, Wis.).

The Clostridial toxin or Clostridial toxin chimeric disclosed in thepresent specification disclosed in the present specification areproduced by the cell in a single-chain form. In order to achieve fullactivity, this single-chain form has to be converted into its di-chainform. As discussed above, this conversion process is achieved bycleaving a protease cleavage site located within the di-chain loopregion of the Clostridial toxin or Clostridial toxin chimeric disclosedin the present specification. This conversion process can be performedusing a standard in vitro proteolytic cleavage assay or in a cell-basedproteolytic cleavage system as described in patent applicationGhanshani, et al., Methods of Intracellular Conversion of Single-ChainProteins into their Di-chain Form, U.S. Pat. No. 8,546,108, which ishereby incorporated by reference in its entirety.

Aspects of the present specification disclose, in part, a compositioncomprising a Clostridial toxin or Clostridial toxin chimeric disclosedin the present specification. In a further aspect, the composition is apharmaceutical acceptable composition. As used herein, the term“pharmaceutically acceptable” refers to any molecular entity orcomposition that does not produce an adverse, allergic, or otheruntoward or unwanted reaction when administered to an individual. Asused herein, the term “pharmaceutically acceptable composition” issynonymous with “pharmaceutical composition” and refers to atherapeutically effective concentration of an active ingredient, suchas, e.g., any of the Clostridial toxins or Clostridial toxin chimerasdisclosed in the present specification. A pharmaceutical compositioncomprising a Clostridial toxin or Clostridial toxin chimeric is usefulfor medical and veterinary applications. A pharmaceutical compositionmay be administered to a patient alone, or in combination with othersupplementary active ingredients, agents, drugs or hormones. Thepharmaceutical compositions may be manufactured using any of a varietyof processes, including, without limitation, conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosageform suitable for administration.

It is also envisioned that a pharmaceutical composition comprising aClostridial toxin or Clostridial toxin chimeric disclosed in the presentspecification can optionally include a pharmaceutically acceptablecarriers that facilitate processing of an active ingredient intopharmaceutically acceptable compositions. As used herein, the term“pharmacologically acceptable carrier” is synonymous with“pharmacological carrier” and refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such acarrier generally is mixed with an active compound or is permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active ingredients can besoluble or can be delivered as a suspension in the desired carrier ordiluent. Any of a variety of pharmaceutically acceptable carriers can beused including, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like;solvents; dispersion media; coatings; antibacterial and antifungalagents; isotonic and absorption delaying agents; or any other inactiveingredient. Selection of a pharmacologically acceptable carrier candepend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activeingredient, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7^(th) ed. 1999); REMINGTON: THE SCIENCE ANDPRACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams &Wilkins, 20^(th) ed. 2000); GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASISOF THERAPEUTICS (Joel G. Hardman et al., eds., McGraw-Hill Professional,10^(th) ed. 2001); and HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (Raymond C.Rowe et al., APhA Publications, 4^(th) edition 2003). These protocolsare routine procedures and any modifications are well within the scopeof one skilled in the art and from the teaching herein.

It is further envisioned that a pharmaceutical composition disclosed inthe present specification can optionally include, without limitation,other pharmaceutically acceptable components (or pharmaceuticalcomponents), including, without limitation, buffers, preservatives,tonicity adjusters, salts, antioxidants, osmolality adjusting agents,physiological substances, pharmacological substances, bulking agents,emulsifying agents, wetting agents, sweetening or flavoring agents, andthe like. Various buffers and refers to for adjusting pH can be used toprepare a pharmaceutical composition disclosed in the presentspecification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate, a stabilized oxy chloro composition, such as, e.g., PURITE® andchelants, such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. The pharmaceuticalcomposition may be provided as a salt and can be formed with manydifferent acids, including, but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, and succinic. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. It is understood that these and other substances knownin the art of pharmacology can be included in a pharmaceuticalcomposition useful in the specification.

In an embodiment, a composition comprises a Clostridial toxin orClostridial toxin chimeric disclosed in the present specification. In anaspect of this embodiment, the composition is a pharmaceuticalcomposition comprising a Clostridial toxin or Clostridial toxin chimericdisclosed in the present specification. In aspects of this embodiment, apharmaceutical composition comprising Clostridial toxin or Clostridialtoxin chimeric disclosed in the present specification further comprisesa pharmacological carrier, a pharmaceutical component, or both apharmacological carrier and a pharmaceutical component. In other aspectsof this embodiment, a pharmaceutical composition comprising aClostridial toxin or Clostridial toxin chimeric disclosed in the presentspecification further comprises at least one pharmacological carrier, atleast one pharmaceutical component, or at least one pharmacologicalcarrier and at least one pharmaceutical component.

Aspects of the present specification can also be described as follows:

-   1. A Clostridial toxin comprising at least one inactivation cleavage    site located within an inactivation cleavage site region, wherein    the inactivation cleavage site region is located in the    translocation domain and/or the H_(CN) binding subdomain, wherein    the at least one inactivation cleavage site comprises a dual    Thrombin-Thrombin site, a Factor Xa site, a dual Factor Xa-Thrombin    site, and/or a MMP-9 site.-   2. A Clostridial toxin comprising a Clostridial toxin enzymatic    domain, a Clostridial toxin translocation domain, a Clostridial    toxin binding domain, a di-chain loop region, an exogenous protease    cleavage site, and at least two inactivation cleavage sites located    within an inactivation cleavage site region; wherein the exogenous    protease cleavage site located within the di-chain loop region.-   3. A Clostridial toxin of aspect 2, wherein the inactivation    cleavage sites comprise a dual Thrombin-Thrombin site and/or a dual    Factor Xa-Thrombin site.-   4. A Clostridial toxin chimeric comprising a Clostridial toxin    enzymatic domain, a Clostridial toxin translocation domain, a    non-Clostridial toxin binding domain, and an inactivation cleavage    located within an inactivation cleavage site region, wherein the    inactivation cleavage site region is located in the translocation    domain and/or the H_(CN) binding subdomain.-   5. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 462-496 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 458-492 of SEQ ID NO: 3; amino    acids 464-487 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 463-496 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 458-491 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 434-467 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 453-486 of SEQ ID NO: 18, SEQ ID    NO: 19, and/or SEQ ID NO: 20; amino acids 458-491 of SEQ ID NO: 21;    amino acids 443-476 of SEQ ID NO: 23; and/or amino acids 434-467 of    SEQ ID NO: 24 and/or SEQ ID NO: 25.-   6. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 618-634 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 614-630 of SEQ ID NO: 3; amino    acids 605-621 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 613-629 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 609-625 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 587-603 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 604-620 of SEQ ID NO: 18; amino    acids 605-621 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    610-626 of SEQ ID NO: 21; amino acids 596-612 of SEQ ID NO: 23;    and/or amino acids 587-603 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   7. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 638-651 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 634-647 of SEQ ID NO: 3; amino    acids 625-638 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 633-646 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 629-642 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 607-620 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 624-637 of SEQ ID NO: 18; amino    acids 625-638 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    630-643 of SEQ ID NO: 21; amino acids 616-629 of SEQ ID NO: 23;    and/or amino acids 607-620 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   8. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 665-687 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 661-683 of SEQ ID NO: 3; amino    acids 652-674 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 660-682 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 656-678 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 634-659 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 651-676 of SEQ ID NO: 18; amino    acids 652-677 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    657-679 of SEQ ID NO: 21; amino acids 643-668 of SEQ ID NO: 23;    and/or amino acids 634-659 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   9. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 752-765 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 748-761 of SEQ ID NO: 3; amino    acids 739-752 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 747-760 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 743-756 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 724-739 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 741-756 of SEQ ID NO: 18; amino    acids 742-757 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    744-757 of SEQ ID NO: 21; amino acids 733-748 of SEQ ID NO: 23;    and/or amino acids 724-739 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   10. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 826-835 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 824-831 of SEQ ID NO: 3; amino    acids 813-824 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 821-830 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 817-826 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 800-809 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 817-826 of SEQ ID NO: 18; amino    acids 818-827 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    818-827 of SEQ ID NO: 21; amino acids 809-819 of SEQ ID NO: 23;    and/or amino acids 800-809 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   11. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 844-863 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 840-859 of SEQ ID NO: 3;; amino    acids 831-850 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 839-858 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 835-854 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 818-837 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 835-854 of SEQ ID NO: 18; amino    acids 836-855 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    836-855 of SEQ ID NO: 21; amino acids 828-847 of SEQ ID NO: 23;    and/or amino acids 818-837 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   12. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-4, wherein the inactivation cleavage site region comprises    amino acids 871-895 of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4    and/or SEQ ID NO: 5; amino acids 867-891 of SEQ ID NO: 3; amino    acids 858-882 of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID    NO: 9, and/or SEQ ID NO: 10; amino acids 866-890 of SEQ ID NO: 11    and/or SEQ ID NO: 12; amino acids 862-886 of SEQ ID NO: 13 and/or    SEQ ID NO: 14; amino acids 845-869 of SEQ ID NO: 15, SEQ ID NO: 16,    and/or SEQ ID NO: 17; amino acids 862-886 of SEQ ID NO: 18; amino    acids 863-887 of SEQ ID NO: 19 and/or SEQ ID NO: 20; amino acids    863-887 of SEQ ID NO: 21; amino acids 855-879 of SEQ ID NO: 23;    and/or amino acids 845-869 of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   13. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-12, wherein the Clostridial toxin enzymatic domain    comprises a BoNT/A enzymatic domain, a BoNT/B enzymatic domain, a    BoNT/C1 enzymatic domain, a BoNT/D enzymatic domain, a BoNT/E    enzymatic domain, a BoNT/F enzymatic domain, a BoNT/G enzymatic    domain, a BaNT enzymatic domain, and/or a BuNT enzymatic domain.-   14. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-13, wherein the inactivation cleavage site comprises    Thrombin cleavage sites, Plasmin cleavage sites, Coagulation Factor    VIIa cleavage sites, Coagulation Factor IXa cleavage sites,    Coagulation Factor Xa cleavage sites, Coagulation Factor XIa    cleavage sites, Coagulation Factor XIIa cleavage sites, plasma    kallikrein cleavage sites, protease-activated G protein-coupled    receptor-1 (PAR1) cleavage sites, PAR2 cleavage sites, PAR3 cleavage    sites, PAR4 cleavage sites, Matrix Metalloproteinase-2 (MMP-2)    cleavage sites, Matrix Metalloproteinase-9 (MMP-9) cleavage sites,    Furin cleavage sites, urokinase-type Plasminogen activator (uPA)    cleavage sites, tissue-type Plasminogen activator (tPA) cleavage    sites, Tryptase-ε cleavage sites, Mouse mast cell protease-7    (mMCP-7) cleavage sites, endothelin-converting enzyme-1 (ECE-1)    cleavage sites, Kell blood group cleavage sites, DPPIV cleavage    sites, ADAM metallopeptidase with thrombospondin type 1 motif-13    (ADAMTS13) cleavage sites, and/or Cathepsin L cleavage sites.-   15. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-14, wherein the Clostridial toxin translocation domain    comprises a BoNT/A translocation domain, a BoNT/B translocation    domain, a BoNT/C1 translocation domain, a BoNT/D translocation    domain, a BoNT/E translocation domain, a BoNT/F translocation    domain, a BoNT/G translocation domain, a TeNT translocation domain,    a BaNT translocation domain, and/or a BuNT translocation domain.-   16. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-15, wherein the inactivation cleavage site comprises a    dual Thrombin-Thrombin site, a Factor Xa site, a dual Factor    Xa-Thrombin site, and/or a MMP-9 site.-   17. The Clostridial toxin and/or Clostridial toxin chimeric of    aspects 1-16, wherein the, a non-Clostridial toxin binding domain,    comprises a opioid binding domain, a tachykinin binding domain, a    melanocortin binding domain, a galanin binding domain, a granin    binding domain, a Neuropeptide Y related peptide binding domain, a    neurohormone binding domain, a neuroregulatory cytokine binding    domain, a kinin peptide binding domain, a growth factor binding    domain, and/or a glucagon like hormone binding domain.-   18. A BoNT/A comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   19. A Clostridial toxin comprising a BoNT/A enzymatic domain, a    BoNT/A translocation domain, a BoNT/A binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   20. A Clostridial toxin comprising a BoNT/A enzymatic domain, a    BoNT/A translocation domain, a BoNT/A binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   21. A Clostridial toxin comprising a BoNT/A enzymatic domain, a    BoNT/A translocation domain, a non-Clostridial toxin binding domain,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   22. A Clostridial toxin comprising a BoNT/A enzymatic domain, a    BoNT/A translocation domain, a non-Clostridial toxin binding domain,    an exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   23. The toxin and/or chimeric of aspects 18-22, wherein the    inactivation cleavage site region comprises amino acids 462-496,    618-634, 638-651, 665-687, 752-765, 826-835, 844-863, and/or 871-895    of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 5,    and/or amino acids 458-492, 614-630, 634-647, 665-687, 748-761,    822-831, 840-859, and/or 867-891 of SEQ ID NO: 3.-   24. A BoNT/B comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   25. A Clostridial toxin comprising a BoNT/B enzymatic domain, a    BoNT/B translocation domain, a BoNT/B binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   26. A Clostridial toxin comprising a BoNT/B enzymatic domain, a    BoNT/B translocation domain, a BoNT/B binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   27. A Clostridial toxin comprising a BoNT/B enzymatic domain, a    BoNT/B translocation domain, a non-Clostridial toxin binding domain,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   28. A Clostridial toxin comprising a BoNT/B enzymatic domain, a    BoNT/B translocation domain, a non-Clostridial toxin binding domain,    an exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   29. The toxin and/or chimeric of aspects 24-28, wherein the    inactivation cleavage site region comprises amino acids 464-487,    605-621, 625-638, 652-674, 739-752, 813-824, 831-850, and/or 858-882    of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and/or    SEQ ID NO: 10.-   30. A BoNT/C1 comprising an inactivation cleavage site located    within an inactivation cleavage site region, wherein the    inactivation cleavage site region is located in the translocation    domain and/or the H_(CN) binding subdomain.-   31. A Clostridial toxin comprising a BoNT/C1 enzymatic domain, a    BoNT/C1 translocation domain, a BoNT/C1 binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   32. A Clostridial toxin comprising a BoNT/C1 enzymatic domain, a    BoNT/C1 translocation domain, a BoNT/C1 binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   33. A Clostridial toxin comprising a BoNT/C1 enzymatic domain, a    BoNT/C1 translocation domain, a non-Clostridial toxin binding    domain, and an inactivation cleavage site located within an    inactivation cleavage site region, wherein the inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   34. A Clostridial toxin comprising a BoNT/C1 enzymatic domain, a    BoNT/C1 translocation domain, a non-Clostridial toxin binding    domain, an exogenous protease cleavage site, a di-chain loop region,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the exogenous protease cleavage site    is located within the di-chain loop region; the wherein inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   35. The toxin and/or chimeric of aspects 30-34, wherein the    inactivation cleavage site region comprises amino acids 463-496,    613-629, 633-646, 660-682, 747-760, 821-830, 839-858, and/or 866-890    of SEQ ID NO: 11 and/or SEQ ID NO: 12.-   36. A BoNT/D comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   37. A Clostridial toxin comprising a BoNT/D enzymatic domain, a    BoNT/D translocation domain, a BoNT/D binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   38. A Clostridial toxin comprising a BoNT/D enzymatic domain, a    BoNT/D translocation domain, a BoNT/D binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   39. A Clostridial toxin comprising a BoNT/D enzymatic domain, a    BoNT/D translocation domain, a non-Clostridial toxin binding domain,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   40. A Clostridial toxin comprising a BoNT/D enzymatic domain, a    BoNT/D translocation domain, a non-Clostridial toxin binding domain,    an exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   41. The toxin and/or chimeric of aspects 36-40, wherein the    inactivation cleavage site region comprises amino acids 458-491,    609-625, 629-642, 656-678, 743-756, 817-826, 835-854, and/or 862-886    of SEQ ID NO: 13 and/or SEQ ID NO: 14.-   42. A BoNT/E comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   43. A Clostridial toxin comprising a BoNT/E enzymatic domain, a    BoNT/E translocation domain, a BoNT/E binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   44. A Clostridial toxin comprising a BoNT/E enzymatic domain, a    BoNT/E translocation domain, a BoNT/E binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   45. A Clostridial toxin comprising a BoNT/E enzymatic domain, a    BoNT/E translocation domain, a non-Clostridial toxin binding domain,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   46. A Clostridial toxin comprising a BoNT/E enzymatic domain, a    BoNT/E translocation domain, a non-Clostridial toxin binding domain,    an exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   47. The toxin and/or chimeric of aspects 42-46, wherein the    inactivation cleavage site region comprises amino acids 434-467,    587-603, 607-620, 634-659, 724-739, 800-809, 818-837, and/or 845-869    of SEQ ID NO: 15, SEQ ID NO: 16, and/or SEQ ID NO: 17.-   48. A BoNT/F comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   49. A Clostridial toxin comprising a BoNT/F enzymatic domain, a    BoNT/F translocation domain, a BoNT/F binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   50. A Clostridial toxin comprising a BoNT/F enzymatic domain, a    BoNT/F translocation domain, a BoNT/F binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   51. A Clostridial toxin comprising a BoNT/F enzymatic domain, a    BoNT/F translocation domain, a non-Clostridial toxin binding domain,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   52. A Clostridial toxin comprising a BoNT/F enzymatic domain, a    BoNT/F translocation domain, a non-Clostridial toxin binding domain,    an exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   53. The toxin and/or chimeric of aspects 48-52, wherein the    inactivation cleavage site region comprises amino acids 453-486,    604-620, 624-637, 651-676, 741-756, 817-826, 835-854, and/or 862-886    of SEQ ID NO: 18; and/or amino acids 453-486, 605-621, 625-638,    652-677, 742-757, 818-827, 836-855, and/or 863-887 of SEQ ID NO: 19    and/or SEQ ID NO: 20.-   54. A BoNT/G comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   55. A Clostridial toxin comprising a BoNT/G enzymatic domain, a    BoNT/G translocation domain, a BoNT/G binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   56. A Clostridial toxin comprising a BoNT/G enzymatic domain, a    BoNT/G translocation domain, a BoNT/G binding domain, an exogenous    protease cleavage site, a di-chain loop region, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the exogenous protease cleavage site is located within the    di-chain loop region; the wherein inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   57. A Clostridial toxin comprising a BoNT/G enzymatic domain, a    BoNT/G translocation domain, a non-Clostridial toxin binding domain,    and an inactivation cleavage site located within an inactivation    cleavage site region, wherein the inactivation cleavage site region    is located in the translocation domain and/or the H_(CN) binding    subdomain.-   58. A Clostridial toxin comprising a BoNT/G enzymatic domain, a    BoNT/G translocation domain, a non-Clostridial toxin binding domain,    an exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   59. The toxin and/or chimeric of aspects 54-58, wherein the    inactivation cleavage site region comprises amino acids 458-491,    610-626, 630-643, 657-679, 744-757, 818-827, 836-855, and/or 863-887    of SEQ ID NO: 21.-   60. A BaNT comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   61. A Clostridial toxin comprising a BaNT enzymatic domain, a BaNT    translocation domain, a BaNT binding domain, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the inactivation cleavage site region is located in the    translocation domain and/or the H_(CN) binding subdomain.-   62. A Clostridial toxin comprising a BaNT enzymatic domain, a BaNT    translocation domain, a BaNT binding domain, an exogenous protease    cleavage site, a di-chain loop region, and an inactivation cleavage    site located within an inactivation cleavage site region, wherein    the exogenous protease cleavage site is located within the di-chain    loop region; the wherein inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   63. A Clostridial toxin comprising a BaNT enzymatic domain, a BaNT    translocation domain, a non-Clostridial toxin binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   64. A Clostridial toxin comprising a BaNT enzymatic domain, a BaNT    translocation domain, a non-Clostridial toxin binding domain, an    exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   65. The toxin and/or chimeric of aspects 60-64, wherein the    inactivation cleavage site region comprises amino acids 443-476,    596-612, 616-629, 643-668, 733-748, 809-819, 828-847, and/or 855-879    of SEQ ID NO: 23.-   66. A BuNT comprising an inactivation cleavage site located within    an inactivation cleavage site region, wherein the inactivation    cleavage site region is located in the translocation domain and/or    the H_(CN) binding subdomain.-   67. A Clostridial toxin comprising a BuNT enzymatic domain, a BuNT    translocation domain, a BuNT binding domain, and an inactivation    cleavage site located within an inactivation cleavage site region,    wherein the inactivation cleavage site region is located in the    translocation domain and/or the H_(CN) binding subdomain.-   68. A Clostridial toxin comprising a BuNT enzymatic domain, a BuNT    translocation domain, a BuNT binding domain, an exogenous protease    cleavage site, a di-chain loop region, and an inactivation cleavage    site located within an inactivation cleavage site region, wherein    the exogenous protease cleavage site is located within the di-chain    loop region; the wherein inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   69. A Clostridial toxin comprising a BuNT enzymatic domain, a BuNT    translocation domain, a non-Clostridial toxin binding domain, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the inactivation cleavage site region is    located in the translocation domain and/or the H_(CN) binding    subdomain.-   70. A Clostridial toxin comprising a BuNT enzymatic domain, a BuNT    translocation domain, a non-Clostridial toxin binding domain, an    exogenous protease cleavage site, a di-chain loop region, and an    inactivation cleavage site located within an inactivation cleavage    site region, wherein the exogenous protease cleavage site is located    within the di-chain loop region; the wherein inactivation cleavage    site region is located in the translocation domain and/or the H_(CN)    binding subdomain.-   71. The toxin and/or chimeric of aspects 66-70, wherein the    inactivation cleavage site region comprises amino acids 434-467,    587-603, 607-620, 634-659, 724-739, 800-809, 818-837, and/or 845-869    of SEQ ID NO: 24 and/or SEQ ID NO: 25.-   72. The toxin and/or chimeric of aspects 1-71, wherein the    Clostridial toxin and/or Clostridial toxin chimeric comprising an    inactivation cleavage site has a safety margin that is greater    relative to the same and/or similar to the Clostridial toxin and/or    Clostridial toxin chimeric, but without the inactivation cleavage    site.-   73. The toxin and/or chimeric of aspect 72, wherein the Clostridial    toxin and/or Clostridial toxin chimeric comprising an inactivation    cleavage site has a safety margin that is greater than at least 10%,    at least 20%, at least 30%, at least 40%, at least 50%, at least    60%, at least 70%, at least 80%, at least 90%, at least 100%, 110%,    at least 120%, at least 130%, at least 140%, at least 150%, at least    160%, at least 170%, at least 180%, at least 190%, at least 200%,    210%, at least 220%, at least 230%, at least 240%, at least 250%, at    least 260%, at least 270%, at least 280%, at least 290%, and/or at    least 300%, relative to the same and/or similar Clostridial toxin    and/or Clostridial toxin chimeric, but without the inactivation    cleavage site, and/or    -   wherein Clostridial toxin and/or Clostridial toxin chimeric        comprising an inactivation cleavage site has a safety margin        that is greater than at most 10%, at most 20%, at most 30%, at        most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at        most 90%, at most 100%, 110%, at most 120%, at most 130%, at        most 140%, at most 150%, at most 160%, at most 170%, at most        180%, at most 190%, at most 200%, 210%, at most 220%, at most        230%, at most 240%, at most 250%, at most 260%, at most 270%, at        most 280%, at most 290%, and/or at most 300%, relative to the        same and/or similar Clostridial toxin and/or Clostridial toxin        chimeric, but without the inactivation cleavage site, and/or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprising an inactivation cleavage site has a safety margin        that is greater by about 10% to about 300%, about 20% to about        300%, about 30% to about 300%, about 40% to about 300%, about        50% to about 300%, about 60% to about 300%, about 70% to about        300%, about 80% to about 300%, about 90% to about 300%, and/or        about 100% to about 300%, relative to the same and/or similar        Clostridial toxin and/or Clostridial toxin chimeric, but without        the inactivation cleavage site, or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprising an inactivation cleavage site has a safety margin        that is greater than at least 1-fold, at least 1-fold, at least        3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at        least 7-fold, at least 8-fold, at least 9-fold, and/or at least        10-fold, relative to the same and/or similar Clostridial toxin        and/or Clostridial toxin chimeric, but without the inactivation        cleavage site, and/or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprising an inactivation cleavage site has a safety margin        that is greater than at least 1-fold, at most 1-fold, at most        3-fold, at most 4-fold, at most 5-fold, at most 6-fold, at most        7-fold, at most 8-fold, at most 9-fold, and/or at most 10-fold,        relative to the same and/or similar Clostridial toxin and/or        Clostridial toxin chimeric, but without the inactivation        cleavage site, and/or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprising an inactivation cleavage site has a safety margin        that is greater by about 1-fold to about 10-fold, about 1-fold        to about 9-fold, about 1-fold to about 8-fold, about 1-fold to        about 7-fold, about 1-fold to about 6-fold, about 1-fold to        about 5-fold, about 2-fold to about 10-fold, about 2-fold to        about 9-fold, about 2-fold to about 8-fold, about 2-fold to        about 7-fold, about 2-fold to about 6-fold, and/or about 2-fold        to about 5-fold.-   74. The toxin and/or chimeric of aspects 1-73, wherein the addition    of the inactivation cleavage site increases the safety margin of the    Clostridial toxin and/or Clostridial toxin chimeric relative to the    same and/or similar Clostridial toxin and/or Clostridial toxin    chimeric, but without the additional inactivation cleavage site.-   75. The toxin and/or chimeric of aspect 74, wherein the Clostridial    toxin and/or Clostridial toxin chimeric comprises the addition of an    inactivation cleavage site that increases the safety margin of the    Clostridial toxin and/or Clostridial toxin chimeric relative to the    same and/or similar Clostridial toxin and/or Clostridial toxin    chimeric, but without the additional inactivation cleavage site by    at least 10%, at least 20%, at least 30%, at least 40%, at least    50%, at least 60%, at least 70%, at least 80%, at least 90%, at    least 100%, 110%, at least 120%, at least 130%, at least 140%, at    least 150%, at least 160%, at least 170%, at least 180%, at least    190%, at least 200%, 210%, at least 220%, at least 230%, at least    240%, at least 250%, at least 260%, at least 270%, at least 280%, at    least 290%, and/or at least 300%, or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprises the addition of an inactivation cleavage site that        increases the safety margin of the Clostridial toxin and/or        Clostridial toxin chimeric relative to the same and/or similar        Clostridial toxin and/or Clostridial toxin chimeric, but without        the additional inactivation cleavage site by at most 10%, at        most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at        most 70%, at most 80%, at most 90%, at most 100%, 110%, at most        120%, at most 130%, at most 140%, at most 150%, at most 160%, at        most 170%, at most 180%, at most 190%, at most 200%, 210%, at        most 220%, at most 230%, at most 240%, at most 250%, at most        260%, at most 270%, at most 280%, at most 290%, and/or at most        300%, or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprises the addition of an inactivation cleavage site that        increases the safety margin of the Clostridial toxin and/or        Clostridial toxin chimeric relative to the same and/or similar        Clostridial toxin and/or Clostridial toxin chimeric, but without        the additional inactivation cleavage site by about 10% to about        300%, about 20% to about 300%, about 30% to about 300%, about        40% to about 300%, about 50% to about 300%, about 60% to about        300%, about 70% to about 300%, about 80% to about 300%, about        90% to about 300%, and/or about 100% to about 300%, or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprises the addition of an inactivation cleavage site that        increases the safety margin of the Clostridial toxin and/or        Clostridial toxin chimeric relative to the same and/or similar        Clostridial toxin and/or Clostridial toxin chimeric, but without        the additional inactivation cleavage site by at least 1-fold, at        least 1-fold, at least 3-fold, at least 4-fold, at least 5-fold,        at least 6-fold, at least 7-fold, at least 8-fold, at least        9-fold, and/or at least 10-fold, and/or wherein the Clostridial        toxin and/or Clostridial toxin chimeric comprises the addition        of an inactivation cleavage site that increases the safety        margin of the Clostridial toxin and/or Clostridial toxin        chimeric relative to the same and/or similar Clostridial toxin        and/or Clostridial toxin chimeric, but without the additional        inactivation cleavage site by, e.g., at most 1-fold, at most        3-fold, at most 4-fold, at most 5-fold, at most 6-fold, at most        7-fold, at most 8-fold, at most 9-fold, and/or at most 10-fold,        and/or    -   wherein the Clostridial toxin and/or Clostridial toxin chimeric        comprises the addition of an inactivation cleavage site that        increases the safety margin of the Clostridial toxin and/or        Clostridial toxin chimeric relative to the same and/or similar        Clostridial toxin and/or Clostridial toxin chimeric, but without        the additional inactivation cleavage site by about 1-fold to        about 10-fold, about 1-fold to about 9-fold, about 1-fold to        about 8-fold, about 1-fold to about 7-fold, about 1-fold to        about 6-fold, about 1-fold to about 5-fold, about 2-fold to        about 10-fold, about 2-fold to about 9-fold, about 2-fold to        about 8-fold, about 2-fold to about 7-fold, about 2-fold to        about 6-fold, and/or about 2-fold to about 5-fold.-   76. A polynucleotide molecule encoding a toxin and/or chimeric    according to any one of aspects 1-75.-   77. The polynucleotide molecule, wherein the molecule comprises SEQ    ID NO: 530, SEQ ID NO: 532, SEQ ID NO: 534, and/or SEQ ID NO: 536.-   78. A method of producing a Clostridial toxin and/or Clostridial    toxin chimeric comprising the step of expressing in a cell a    polynucleotide molecule according to aspect 76 and/or 77, wherein    expression from the polynucleotide molecule produces the encoded    Clostridial toxin and/or Clostridial toxin chimeric.-   79. A method of producing a modified Clostridial toxin comprising    the steps of:    -   a. introducing into a cell a polynucleotide molecule according        to aspect 76 and/or 77; and    -   b. expressing the polynucleotide molecule, wherein expression        from the polynucleotide molecule produces the encoded        Clostridial toxin and/or Clostridial toxin chimeric.-   80. A Clostridial toxin comprising SEQ ID NO: 531, SEQ ID NO: 533,    SEQ ID NO: 535, and/or SEQ ID NO: 537.-   81. A Clostridial toxin comprising SEQ ID NO: 531.-   82. A Clostridial toxin comprising SEQ ID NO: 533.-   83. A Clostridial toxin comprising SEQ ID NO: 535.-   84. A Clostridial toxin comprising SEQ ID NO: 537.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofdisclosed embodiments and are in no way intended to limit any of theembodiments disclosed in the present specification.

Example 1 Identification of Inactivation Cleavage Site Regions

This example illustrates how to identify regions within a Clostridialtoxin or Clostridial toxin chimeric suitable for modifying the toxin tocomprise an inactivation cleavage site and how to make a Clostridialtoxin or Clostridial toxin chimeric comprising an inactivation cleavagesite.

To identify a location or locations in the protein structure suitable asa potential inactivation cleavage site region, the three-dimensionalstructure of a BoNT/A was initially analyzed by computer software toidentify surface exposed loops or extended regions that would be moreaccessible to a protease. Of the regions predicted to be accessible,eight were selected for further analysis: amino acids 462-496 of SEQ IDNO: 1, amino acids 618-634 of SEQ ID NO: 1, amino acids 638-651 of SEQID NO: 1, amino acids 665-687 of SEQ ID NO: 1, amino acids 752-765 ofSEQ ID NO: 1, and amino acids 826-835 of SEQ ID NO: 1, amino acids844-863 of SEQ ID NO: 1, and amino acids 871-895 of SEQ ID NO: 1.

To determine whether a region identified by computer analysis couldfunction as an inactivation cleavage site region, thrombin cleavagesites were genetically engineered into these regions using multi-primermutagenesis and assayed for its ability to be cleaved by thrombin. A 50μL reaction was assembled comprising a primer pool of unidirectionaloligonucleotide primers each containing the desired modification (125 ngof each primer) mixed in different ratios with a DNA template comprisingan expression construct encoding a BoNT/A, such as, e.g., an expressionconstruct comprising SEQ ID NO: 526 encoding SEQ ID NO: 527, or anexpression construct comprising SEQ ID NO: 528 encoding SEQ ID NO: 529,that was hypermethylated with dam methylase. To this mixture was added 5μL of 10×PCR Buffer, 1 μL of deoxyribonucleotides (dNTPs), 1 μL of 2.5units/ΞL PFUULTRA™ High Fidelity DNA polymerase (Stratagene, La Jolla,Calif.), Pfu DNA ligase, ATP, and nuclease-free water to a final volumeof 50 μL. The thermocycler conditions were: 30 cycles of 96° C. for 1minute, 60° C. for 30 seconds, and 68° C. for 20 minutes. Followingthermocycling, 1 μL of DpnI restriction enzyme (Stratagene, La Jolla,Calif.) was added to the reaction and incubated for 1 hour at 37° C. todigest the template DNA and reduce the recovery of wild-type clones. Thedigested reaction mixture was transformed into electro-competent E. coliBL21(DE3) Acella cells (Edge BioSystems, Gaithersburg, Md.) byelectroporation, plated on 1.5% Luria-Bertani agar plates (pH 7.0)containing 50 μg/mL of kanamycin, and placed in a 37° C. incubator forovernight growth. Bacteria containing expression constructs wereidentified as kanamycin resistant colonies. Candidate constructs wereisolated using an alkaline lysis plasmid mini-preparation procedure andanalyzed by sequencing to determine the frequency and identity of themutations incorporated. Table 7 lists each BoNT/A comprising a Thrombincleavage site (BoNT/A-TCS) made and tested in this thrombin scanninganalysis.

TABLE 7 Thrombin Scanning Analysis Ex- Thrombin BoNT/A RegionModification pression Sensitivity Potency 462-496 T482insLVPRGS + ND ND462-496 A489insLVPRGS ++ ++ ND 618-634 E620insLVPRGS + ND ND 638-651M646insLVPRGS −/+ ND ND 665-687 I673insLVPRGS + ND ND 752-765E758insLVPRGS − ND ND 826-835 delR827GT- −/+ ND ND insLVPRGS 844-863T844insLVPRGS +++ + ND 844-863 D848insLVPRGS +++ + ND 844-863Q852insLVPRGS −/+ ND ND 844-863 L862insLVPRGS +++ ++ ND 871-895E868insLVPRGS ND 871-895 delE868YIKNI- ND insLVPRGS 871-895K871insLVPRGS +++ +++ ND 871-895 I873insLVPRGS +++ ++++ ND 871-895delN872IINTS- ND insLVPRGS 871-895 T876insLVPRGS ND 871-895 L879insVPRGSND 871-895 delL879NLRYE- ND insLVPRGS 871-895 N880insLVPRGS +++ ++++4.05 871-895 L881insVPRGS ND 871-895 delL881RYESN- ND insLVPRGS 871-895Y883insLVPRGS ND 871-895 E884insLVPRGS +++ +++ >50 871-895 S885insLVPRGSND 871-895 delH887LIDLS- ND insLVPRGS 871-895 L888insVPRGS ND 871-895D890insLVPRGS ++ ++++ 3.15 871-895 L891insVPRG ND 871-895 delS892RYA- NDinsVPRG 467-496 T482insLVPRGS + ND ND A489insLVPRGS 618-634E620insLVPRGS + ND ND 665-687 I673insLVPRGS 638-651 M646insLVPRGS + NDND 665-687 I673insLVPRGS 825-832 delR827GT- + +++ ND insLVPRGS 871-895K871insLVPRGS 844-863 T844insLVPRGS −/+ ND ND Q852insLVPRGS 844-863Q852insLVPRGS − ND ND L862insLVPRGS 825-832 delR827GT- + ND ND insLVPRGS871-895 K871insLVPRGS 825-832 delR827GT- − ND ND insLVPRGS 871-895K880insLVPRGS 871-895 E868insLVPRGS 1.32 I873insLVPRGS 871-895delE868YIKNI- 0.86 insLVPRGS delL881RYESN- insLVPRGS 871-895I873insLVPRGS 1.32 E884insLVPRGS 871-895 L881insVPRGS +++ ++++ 4.20L891insVPRG Control Backbone +++ − Yes Protease sensitivity: +, lessthan 25% of toxin proteolyzed within about 1 to about 4 hours; ++, from25% to 50% of toxin proteolyzed within about 1 to about 4 hours; +++,from 51% to 75% of toxin proteolyzed within about 1 to about 4 hours;+++, more than 75% of toxin proteolyzed within about 1 to about 4 hours.BoNT/A potency is calculated by dividing the EC₅₀ value of the toxininto the EC₅₀ value of the backbone control. ND is not determined.

To determine the expression level of soluble protein for eachBoNT/A-TCS, an expression construct comprising each BoNT/A-TCS wasexpressed, purified by immobilized metal affinity chromatography andanalyzed by SDS-PAGE analysis. First, using a 96-well plate, 100 μL ofPA-0.5G media containing 50 μg/mL Kanamycin was inoculated with a singlecolony of BL21(DE3) cells harboring the appropriate expression constructand grown at 37° C. with shaking overnight. A 5 μL aliquot from thisstarter culture was used to inoculate 1 mL of ZYP-5052 containing 50μg/mL kanamycin and grown at 37° C. with shaking for 3.5 hours and then22° C. for 16 hours. A 110 μL aliquot of Protein Extraction Reagentcomprising 10× FASTBREAK™ Cell Lysis Reagent (Promega Corp., Madison,Wis.), 250 U/mL Benzonase nuclease (EMD Biosciences-Novagen, Madison,Wis.), and 10× Protease Inhibitor Cocktail III (EMDBiosciences-Calbiochem, Gibbstown, N.J.) was added to each 1 mLexpression culture in a 96-well plate. 75 μL of HISLINK™ resin (PromegaCorp., Madison, Wis.) was next transferred to each well and the mixturewas alternately mixed by pipetting and by shaking at 900 rpm for 30minutes. The lysates were transferred to a filter plate with a 25 μmpore size (Promega Corp., Madison, Wis.), with membranes pre-wetted withwater, and the liquid was removed by vacuum filtration. The resin waswashed three times with 200 μL Wash Buffer comprising 100 mM HEPES (pH7.5), 10 mM imidazole. The protein was eluted by adding 200 μL ElutionBuffer comprising 100 mM HEPES (pH 7.5), 500 mM imidazole, incubatingfor 5 minutes and the elute collected by vacuum filtration into a96-well plate.

To perform SDS-PAGE, an equal volume of 2× Laemmli Sample Buffer wasadded to the IMAC purified BoNT/A comprising a thrombin cleavage site,and the mixture incubated at 95° C. for 5 minutes. A 15 μL aliquot wasloaded and separated by MOPS polyacrylamide gel electrophoresis usingNUPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen,Inc, Carlsbad, Calif.) under denaturing, reducing conditions. The gelwas washed and fixed in 10% methanol and 7% acetic acid for 30 minutes.The wash solution was removed and the gel incubated in SYPRO Rubyprotein gel stain solution for 3 hours to overnight at room temperature.The stained gel was destained in 10% methanol and 7% acetic acid for 30minutes. The destained gel was visualized with a Fluoro-S-Max digitalimager (Bio-Rad).

The results of the expression analysis are given in Table 7. In general,toxins harboring an inserted thrombin cleavage site in the inactivationregions comprising amino acids 462-496 of SEQ ID NO: 1, amino acids844-863, or amino acids 871-895 of SEQ ID NO: 1 were expressed well. Forexample, toxins comprising A489insLVPRGS was expressed at about 50% thatof a wild-type control construct and toxins comprising D848insLVPRGS orN880insLVPRGS were expressed at, or near, control levels (Table 7).These results reveal that inactivation cleavage site regions locatedwithin the translocation domain and/or the H_(CN) binding subdomaintolerated the modification of regions to include a protease cleavagesite.

To further explore the extent to which the inactivation cleavage siteregions identified could tolerate modifications that introduce aprotease cleavage site, toxins were modified to include thrombincleavage sites throughout the region. For example, toxins comprisingT884insLVPRGS or L862ins LVPRGS were made to examine the inactivationcleavage site region comprising 844-863 of SEQ ID NO: 1. Similarly,toxins comprising E868insLVPRGS, deIE868YIKNI-insLVPRGS,deIN87211NTS-insLVPRGS, T876insLVPRGS, L879insVPRGS,deIL879NLRYE-insLVPRGS, L881 insVPRGS, deIL881 RYESN-insLVPRGS,Y883insLVPRGS, E884insLVPRGS, S885insLVPRGS, deIH887LIDLS-insLVPRGS,L888insLVPRGS, L891 insVPRG, and deIS892RYA-insVPRG were made to examinethe inactivation cleavage site region comprising 871-895 of SEQ IDNO: 1. Both insertion and substitution modifications were made toexamine whether the type of modification had any affect. In general, alltoxins harboring an inserted thrombin cleavage site in theseinactivation regions were expressed at, or near, the levels of awild-type control construct. These results reveal that inactivationcleavage site regions within the translocation domain and/or the H_(CN)binding subdomain can tolerate modifications placed anywhere within aninactivation site region.

Lastly, the ability of an inactivation site region to tolerate thepresence of two or more protease cleavage sites was examined (Table 7).These results indicate that inactivation cleavage site regions withinthe translocation domain and/or the H_(CN) binding subdomain cantolerate modifications placing two or more protease cleavage siteswithin an inactivation site region.

To determine whether a BoNT/A comprising a thrombin cleavage site couldbe cleaved by thrombin, an in vitro thrombin cleavage assay wasperformed. 5 μg of each purified BoNT/A-TCS was incubated with 1 U ofThrombin (Novagen) at 23° C. for 1 hour, 3 hours, and 18.5 hours. Azero-enzyme control was also set up in parallel for each BoNT/A-TCS.Samples were taken at each time point and quenched with SDS-LoadingBuffer including DTT and analyzed by SDS-PAGE as described above.

The results of the expression analysis are given in Table 7. In general,modification of an inactivation cleavage site region comprising aminoacids 467-496, 844-863, or 871-895 of SEQ ID NO: 1 to include a proteasecleavage site resulted in a toxin that was susceptible to proteolyticcleavage by the appropriate protease.

To determine whether a BoNT/A comprising a thrombin cleavage sitemaintained its potency, a BoNT/A activity assay was performed using acell-based activity assay. To conduct a cell-based activity assay, about1.2×10⁶ Neuro-2a or SiMa cells were plated into the wells of 24-welltissue culture plates containing 1 mL of serum-free medium containingMinimum Essential Medium, 2 mM GLUTAMAX™ I with Earle's salts, 1×B27supplement, 1×N2 supplement, 0.1 mM Non-Essential Amino Acids, 10 mMHEPES and 25 μg/mL GT1b. The cells were incubated in a 37° C. incubatorunder 5% carbon dioxide until the cells differentiated, as assessed bystandard and routine morphological criteria, such as growth arrest andneurite extension (approximately 3 days). The media was aspirated fromeach well and replaced with either 1) fresh media containing no toxin(untreated cell line) or 2) fresh media containing 1 nM of a BoNT/Acomplex (treated cell line). After an overnight incubation, the cellswere washed by aspirating the media and rinsing each well with 200 μL of1×PBS. To harvest the cells, the 1×PBS was aspirated, the cells werelysed by adding 50 μl of 2×SDS Loading Buffer, the lysate wastransferred to a clean test tube and the sample was heated to 95° C. for5 minutes.

To detect for the presence of cleaved SNAP-25 products, an aliquot fromeach harvested sample was analyzed by Western blot. In this analysis, a12 μl aliquot of the harvested sample was separated by MOPSpolyacrylamide gel electrophoresis using NUPAGE® Novex 12% Bis-Trisprecast polyacrylamide gels (Invitrogen Inc., Carlsbad, Calif.) underdenaturing, reducing conditions. Separated peptides were transferredfrom the gel onto polyvinylidene fluoride (PVDF) membranes (InvitrogenInc., Carlsbad, Calif.) by Western blotting using a TRANS-BLOT® SDsemi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories,Hercules, Calif.). PVDF membranes were blocked by incubating at roomtemperature for 2 hours in a solution containing Tris-Buffered Saline(TBS) (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl)(pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride),0.1% TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate), 2% BovineSerum Albumin (BSA), 5% nonfat dry milk. Blocked membranes wereincubated at 4° C. for overnight in TBS, 0.1% TWEEN-20® (polyoxyethylene(20) sorbitan monolaurate), 2% BSA, and 5% nonfat dry milk containingeither 1) a 1:5,000 dilution of an α-SNAP-25 mouse monoclonal antibodyas the primary antibody (SMI-81; Sternberger Monoclonals Inc.,Lutherville, Md.); or 2) a 1:5,000 dilution of S9684 α-SNAP-25 rabbitpolyclonal antiserum as the primary antibody (Sigma, St. Louis, Mo.).Both α-SNAP-25 mouse monoclonal and rabbit polyclonal antibodies candetect both the uncleaved SNAP-25 substrate and the SNAP-25 cleavageproduct, allowing for the assessment of overall SNAP-25 expression ineach cell line and the percent of SNAP-25 cleaved after BoNT/A treatmentas a parameter to assess the amount of BoNT/A uptake. Primary antibodyprobed blots were washed three times for 15 minutes each time in TBS,TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Washed membraneswere incubated at room temperature for 2 hours in TBS, 0.1% TWEEN-20®(polyoxyethylene (20) sorbitan monolaurate), 2% BSA, and 5% nonfat drymilk containing either 1) a 1:10,000 dilution of goat polyclonalanti-mouse immunoglobulin G, heavy and light chains (IgG, H+L) antibodyconjugated to horseradish peroxidase (Zymed, South San Francisco,Calif.) as a secondary antibody; or 2) a 1:10,000 dilution of goatpolyclonal anti-rabbit immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (Zymed, South SanFrancisco, Calif.) as a secondary antibody. Secondary antibody-probedblots were washed three times for 15 minutes each time in TBS, 0.1%TWEEN-20® (polyoxyethylene (20) sorbitan monolaurate). Signal detectionof the labeled SNAP-25 products were visualized using the ECL Plus™Western Blot Detection System (GE Healthcare, Amersham Biosciences,Piscataway, N.J.) and the membrane was imaged and the percent of cleavedquantified with a Typhoon 9410 Variable Mode Imager and Imager Analysissoftware (GE Healthcare, Amersham Biosciences, Piscataway, N.J.). Thechoice of pixel size (100 to 200 pixels) and PMT voltage settings (350to 600, normally 400) depended on the individual blot.

The results of the expression analysis are given in Table 7. In general,modification of an inactivation cleavage site region comprising aminoacids 467-496, 844-863, or 871-895 of SEQ ID NO: 1 to include a proteasecleavage site resulted in a potent toxin that was able to execute theoverall intoxication process.

Taken together, these results indicate that although eight differentinactivation cleavage regions were identified, not all were able tosupport the insertion of a functional thrombin cleavage site. Ingeneral, modification of the inactivation cleavage site regionscomprising amino acids 467-496, 844-863 and 871-895 of SEQ ID NO: 1 toinclude a protease cleavage site resulted in a stably produced toxinthat was able to execute the overall intoxication process and wassensitive to proteolytic cleavage by the appropriate protease.

Because the three-dimensional structure of all Clostridial toxins aresimilar, the corresponding locations in BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F, BoNT/G, TeNT, BaNT, and BuNT are also suitable as inactivationcleavage site regions. Table 5 lists these regions.

Example 2 Protease Cleavage Site Analysis

This example illustrates how to make a Clostridial toxin or Clostridialtoxin chimeric comprising an inactivation cleavage site.

To explore whether protease cleavage sites other than thrombin could beuseful as an inactivation site, toxins comprising many differentprotease cleavage sites were examined.

To make a Clostridial toxin or Clostridial toxin chimeric comprising aninactivation cleavage site, protease cleavage sites were geneticallyengineered into inactivation cleavage site regions using multi-primermutagenesis as described in Example 1. Table 8 lists the expressionconstructs modified to contain a protease cleavage site.

To determine whether a BoNT/A comprising a protease cleavage site couldbe cleaved by its cognate protease, in vitro protease cleavage assayswas performed essentially as described above, but using the appropriateprotease instead of thrombin. Samples were taken at each time point andquenched with SDS-Loading Buffer including DTT, and analyzed by SDS-PAGEas described in Example 1.

The results of the expression analysis are given in Table 7. In general,modification of an inactivation cleavage site region comprising aminoacids 467-496, 844-863, or 871-895 of SEQ ID NO: 1 to include a proteasecleavage site resulted in a toxin that was susceptible to proteolyticcleavage by the appropriate protease.

TABLE 8 Protease Cleavage Site Analysis Protease Protease BoNT/ACleavage Site Region Modification Sensitivity Potency Factor Xa 535E535insG + 2.70 Factor Xa 844-863 L863insIEGR + >50 Factor Xa 871-895K871insIEGR ++ 6.15 Factor Xa 871-895 I873insEGR + 3.97 Factor Xa871-895 L881insIEG ND ND Factor Xa 871-895 E884insIEGR + 2.95 Factor Xa871-895 L891insIEGR ++ ND Factor Xa 1272 E1272insG + ND Factor Xa × 2535 E535insG + ND 1272 E1272insG Factor Xa × 2 871-895 K871insIEGR ++4.35 L891insIEGR Factor Xa × 2 871-895 I873insEGR + 7.63 L891insIEGRFactor Xa × 2 871-895 L881insIEG ++ >50 L891insIEGR Factor Xa 871-895I873insEGR ND ND tPA delS885NHLIDL- insPQRGRSA Factor Xa 871-895I873insEGR + 3.29 Thrombin E884insLVPRG ++++ MMP-2 871-895S885insGPLGMLSQ + 6.55 MMP-2 871-895 delK871NIINTSI- ++ 5.27 insGPLGMLSQMMP-2 871-895 delS885NHLIDLS- ++ 4.76 insGPLGMLSQ MMP-9 871-895K871insGPLGLWAQ ND ND MMP-9 871-895 delK871NIINTSI- + 3.36 insGPLGLWAQMMP-9 871-895 I873insGPLGLWAQ 22.8 MMP-9 871-895 delI874NTSILNL- 37.7insGPLGLWAQ MMP-9 871-895 delL881RYESNHL- ND ND insGPLGLWAQ MMP-9871-895 E884insGPLGLWAQ ND ND MMP-9 871-895 delS885NHLIDLS- + 4.38insGPLGLWAQ MMP-9 871-895 S885insGPLGLWAQ 3.38 MMP-9 871-895L891insGPLGLWAQ 20.61 MMP-9 871-895 delK871NIINTSI- ND ND ThrombininsGPLGLWAQ E884insLVPRG MMP-9 871-895 delK871NIINTSI- 19.62 Factor XainsGPLGLWAQ E884insIEGR u-PA 871-895 delN872IINTSI- + ND insPGSGKSA u-PA871-895 S885insPGSGKSA ++ 3.00 u-PA 871-895 delN886HLIDL- ++ 4.90insPGSGKSA t-PA 871-895 delN872IINTSI- ++ 3.65 insPQRGRSA t-PA 871-895S885insPQRGRSA +++ 3.30 t-PA 871-895 delS885NHLIDL- ++ 4.80 insPQRGRSAThrombin 871-895 I873LVPRGS ND ND tPA delS885NHLIDL- insPQRGRSA Furin871-895 I870insRKKR +++ 6.70 Furin 871-895 delK871NII-insRKKR + 3.50Furin 871-895 L881insRKK + 7.20 Furin 871-895 delY883ES-insKKR + 12.1Furin 871-895 S892RKK + 15.2 Furin × 2 871-895 delK871NII-insRKKR + 12.6delY883ES-insKKR Furin × 2 871-895 delK871NII-insRKKR ++ 6.00 S892RKKFurin × 3 871-895 delK871NII-insRKKR ND ND delY883ES-insKKR S892RKK Kell871-895 L891insAAF + 10.8 Kell 871-895 delI889DL-insAAF + 4.80 Tryptaseε 871-895 K871insIVGGE + 9.45 Tryptase ε 871-895 K871insRIVGGE + 6.48Tryptase ε 871-895 delN886HLIDL- 5.50 insRIVGGE Tryptase ε 871-895delN886HLIDL- ND ND insKIVGGE mMMCP-7 871-895 K871insSLSSRQSP 3.90mMMCP-7 871-895 delN886HLIDLS- 4.80 insLSSRQSP ECE-1 871-895I870insRPPGFSAF + 5.70 ECE-1 871-895 K871insAFA + 3.85 ECE-1 871-895K871insDIIWVNT- + >50 PEHVVPYGLGS ECE-1 871-895 K871insRPKPQQFFGLM ND NDECE-1 871-895 delYES885NHLIDLS- + 9.20 insPKPQQFFGLM ECE-1 871-895E884insKAFA + 2.95 ECE-1 871-895 delS885NHLIDLS- + 3.70 insRPPGFSAFCathespin L 871-895 I870insRGFFYTPK ++++ 10.3 Cathespin L 871-895K871insLR ++++ 2.25 Cathespin L 871-895 K871insFR ++++ 3.05 Cathespin L871-895 K871insLR 12.6 Thrombin L891insLVPRGS PolyArg 844-863 R861insRRND ND PolyArg 871-895 R882insRRR Yes PolyArg 871-895 S885insRRR 2.22PolyArg 871-895 S892insRRR 3.02 PolyArg × 2 844-863 R861insRR ND ND871-895 K871insRKR PolyArg × 2 844-863 R861insRR ND ND 871-895I873insRRRR PolyArg × 2 844-863 R861insRR ND ND 871-895 R882insRRRPolyArg × 2 871-895 K871insRKR 1.92 S885insRRR PolyArg × 2 871-895R882insRRR ND ND S892insRRR Protease sensitivity: +, less than 25% oftoxin proteolysed within about 1 to about 4 hours; ++, from 25% to 50%of toxin proteolyzed within about 1 to about 4 hours; +++, from 51% to75% of toxin proteolyzed within about 1 to about 4 hours; ++++, morethan 75% of toxin proteolyzed within about 1 to about 4 hours. BoNT/Apotency is calculated by dividing the EC₅₀ value of the toxin into theEC₅₀ value of the backbone control. ND is not determined.

To determine whether a BoNT/A comprising a protease cleavage sitemaintained its potency, the cell-based activity assay described abovewas performed (Table 8). In general, toxins comprising a proteasecleavage site that exhibited an EC₅₀ of about 20 or less were deemed toretain enough potency to warranted evaluation using an animal-basedassay.

Example 3 In Vivo Analysis

This example illustrates how to evaluate a Clostridial toxin orClostridial toxin chimeric comprising an inactivation cleavage siteusing an animal-based assay analysis.

Although the cell-based activity assay is a good assessment of whether aClostridial toxin or Clostridial toxin chimeric comprising aninactivation cleavage site can be cleaved by its cognate protease,certain candidates were selected for evaluation in an animal-basedassay.

To test the activity of a Clostridial toxin or Clostridial toxinchimeric comprising an inactivation cleavage site using an animal-basedassay, an in vivo Digit Abduction Score (DAS) assay was initiallyperformed. CD-1 Fe mice were weighed and placed into subsets of 10animals for each discrete DAS assay. Mice were included into aparticular subset based on the following criteria: 1) good health; 2)robust baseline DAS response of 0; 3) inclusion in a median weight rangeof X±2g established for the selected subset and 4) weight greater than17.0 g.

Each mouse was injected using a 30-gauge needle in the gastrocnemiusmuscle of the right hind limb with either 1) 5 μL of 10.0 nM BoNT/Acomprising an inactivation cleavage site (single-dose DAS study); or 2)5 μL of one of seven different doses of BoNT/A comprising aninactivation cleavage site (0.01 nM, 0.04 nM, 0.12 nM, 0.37 nM, 1.11 nM,3.33 nM and 10.0 nM; Full-Dosing DAS study). As a control, thegastrocnemius muscle of the left hind limb was injected with 5 μL of asolution not containing any toxin. Mice were observed for the DASresponse consecutively for the first 4 days. The DAS was read by liftingeach mouse by the tail and precisely observing the injected hind limbs.The abduction or no abduction of the hind digits reveals the effect ofparalysis due to the test toxin injected in the muscle. The digitabduction of the injected hind limb was compared with that of thenon-injected hind limb and scored accordingly. DAS data was analyzed bycalculating the ED₅₀ dose based on peak mean DAS score and AUC (areaunder the curve) in terms of u/Kg and/or ng/Kg. This was accomplished asfollows: 1) the mean peak DAS score for each dose was calculated in eachstudy; 2) any dose that elicited more than five deaths in any study waseliminated from consideration; 3) the highest dose used in a givenindividual study was the lowest dose which elicited an average peak of4.0; 4) the lowest dose used in a given individual study was the highestdose which elicited an average peak of 0; 5) curves were constructed foreach individual study of average peak DAS vs. log (dose); 6) an AUCvalue was calculated for each group of 10 mice of the multiple groups insome studies; 7) curves were constructed for each individual study ofaverage AUC vs. log (dose); 8) an x, y replicate response curve wasconstructed for each set of multiple identical studies; for each testtoxin; 9) dose-response data were analyzed by non-linear regression(non-weighted) using a three-parameter logistic equation (Sigma Plot v8.0; SPSS Science, Chicago, Ill.) using the following equation:y=a/(1+(x/x0)^(b))where y is the response, a is the asymptotic y_(max), b is the slope, xis the dose, and 0 is the ED₅₀ dose, For peak ED₅₀ determinations,Y_(max) was set to 4 (maximum DAS reading on scale). Mean (peak and/orAUC) ED₅₀ values were computed for each eight-dose study performed.

The results indicate that (Table 9). In general, toxins comprising aninactivation cleavage site that exhibited a relative potency of about 10or above were deemed to retain enough potency to warranted evaluation ofits safety margin.

To determine the safety margin of a Clostridial toxin or Clostridialtoxin chimeric comprising an inactivation cleavage site, a mouselethality assay was performed.

To calculate the safety margin of a Clostridial toxin or Clostridialtoxin chimeric comprising an inactivation cleavage site, the LD₅₀ valueobtained from the mouse lethality assay was divided by the EC₅₀ valueobtained from a full-dosing DAS study. A toxin comprising aninactivation cleavage site was deemed to possess enough activity at theinactivation cleavage site if it exhibited a safety margin value ofabout 15 or more.

TABLE 9 Animal-based Assay Analysis Protease Single-Dose Full-DosingLethality Safety Cleavage DAS DAS Assay Margin Site Region ModificationEC₅₀ Relative EC₅₀ Relative LD₅₀ LD₅₀/DAS ED₅₀ Thrombin 871-895I873insLVPGRS 1.08 30.5 ND ND ND ND Thrombin 871-895 L881insVPRGS 0.377.38 ND ND ND ND Thrombin 871-895 E884insLVPRGS 0.16 25.3 0.15 46.7 1.9012.5 Thrombin 871-895 L891insVPRG 0.12 23.3 0.19 36.8 2.74 14.8 Thrombin× 2 871-895 L881insVPRGS 0.25 11.0 0.15 34.5 4.20 26.9 L891insVPRGFactor Xa 871-895 I873insEGR 0.11 46.3 0.10 70.0 2.39 23.0 Factor Xa871-895 I873insEGR 0.09 37.2 0.26 15.3 6.69 26.9 Thrombin E884insLVPRGMMP-2 871-895 delK871NIINTSI- 0.33 10.0 ND ND ND ND insGPLGMLSQ MMP-2871-895 delS885NHLIDLS- 0.10 34.5 ND ND ND ND insGPLGMLSQ MMP-9 871-895delK871NIINTSI- 0.11 29.1 0.16 27.7 5.04 23.9 insGPLGLWAQ MMP-9 871-895delS885NHLIDLS- 0.08 40.8 ND ND ND ND insGPLGLWAQ u-PA 871-895S885insPGSGKSA 0.03 36.6 ND ND ND ND u-PA 871-895 delN886HLIDL- 0.353.52 ND ND ND ND insPGSGKSA t-PA 871-895 delN872IINTSI- 0.04 30.0 ND NDND ND insPQRGRSA t-PA 871-895 S885insPQRGRSA 0.12 10.1 ND ND ND ND t-PA871-895 delS885NHLIDL- 0.08 16.0 0.27 25.9 4.46 17.2 insPQRGRSA Furin871-895 I870insRKKR 0.80 2.68 ND ND ND ND Furin × 2 871-895delK871NII-insRKKR 0.24 8.93 ND ND ND ND delY883ES-insKKR Furin × 2871-895 delK871NII-insRKKR 0.34 6.25 ND ND ND ND S892RKK Tryptase ε871-895 K871insIVGGE 0.14 37.3 ND ND ND ND Tryptase ε 871-895K871insRIVGGE 0.21 10.4 ND ND ND ND Tryptase ε 871-895 delN886HLIDL-0.13 17.2 ND ND ND ND insRIVGGE ECE-1 871-895 E884insKAFA 0.05 43.1 NDND ND ND Cathespin L 871-895 K871insLR 0.10 34.3 ND ND ND ND Cathespin L871-895 K871insFR 0.27 13.0 ND ND ND ND Control — WT 0.05 57.0 0.07 32.40.88 14.2 ND is not determined.

After the DAS analysis, a Clostridial toxin or Clostridial toxinchimeric comprising an inactivation cleavage site was evaluated using amouse lethality assay in order to determine the safety margin bycomparing the ED₅₀ with the LD₅₀.

Although aspects of the present specification have been described withreference to the disclosed embodiments, one skilled in the art willreadily appreciate that the specific examples disclosed are onlyillustrative of these aspects and in no way limit the presentspecification. Various modifications can be made without departing fromthe spirit of the present specification.

The invention claimed is:
 1. An isolated Clostridial toxin comprising SEQ ID NO:
 533. 